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Viruses, Volume 7, Issue 7 (July 2015) – 40 articles

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1819 KiB  
Article
Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage
by María José Benítez-Galeano, Leticia Rubio, Ana Bertalmío, Diego Maeso, Fernando Rivas and Rodney Colina
Viruses 2015, 7(7), 4152-4168; https://doi.org/10.3390/v7072814 - 22 Jul 2015
Cited by 12 | Viewed by 5403
Abstract
Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 [...] Read more.
Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program. Full article
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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Figure 1

Figure 1
<p>Phylogenetic trees for CTV p25 (<b>A</b>); p20 (<b>B</b>) and p23 (<b>C</b>) genes. Colored and highlighted branches represent genotypes: T30 (green), RB (blue), T36 (violet), VT (red), T3 (yellow) and NC (turquoise). Principal node aLRT values are indicated. Reference strains are marked in grey. Uruguayan samples composed by heterogeneous populations are differentially colored.</p> Full article ">Figure 1 Cont.
<p>Phylogenetic trees for CTV p25 (<b>A</b>); p20 (<b>B</b>) and p23 (<b>C</b>) genes. Colored and highlighted branches represent genotypes: T30 (green), RB (blue), T36 (violet), VT (red), T3 (yellow) and NC (turquoise). Principal node aLRT values are indicated. Reference strains are marked in grey. Uruguayan samples composed by heterogeneous populations are differentially colored.</p> Full article ">Figure 2
<p>Evidence of recombination of clone 6 from sample 164 on p25 region between NZ-M16 and T36 isolates (<b>A</b>); Recombination point location detected by GARD tool (<b>B</b>).</p> Full article ">
2919 KiB  
Article
Pigeon RIG-I Function in Innate Immunity against H9N2 IAV and IBDV
by Wenping Xu, Qiang Shao, Yunlong Zang, Qiang Guo, Yongchao Zhang and Zandong Li
Viruses 2015, 7(7), 4131-4151; https://doi.org/10.3390/v7072813 - 22 Jul 2015
Cited by 15 | Viewed by 6844
Abstract
Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and [...] Read more.
Retinoic acid-inducible gene I (RIG-I), a cytosolic pattern recognition receptor (PRR), can sense various RNA viruses, including the avian influenza virus (AIV) and infectious bursal disease virus (IBDV), and trigger the innate immune response. Previous studies have shown that mammalian RIG-I (human and mice) and waterfowl RIG-I (ducks and geese) are essential for type I interferon (IFN) synthesis during AIV infection. Like ducks, pigeons are also susceptible to infection but are ineffective propagators and disseminators of AIVs, i.e., “dead end” hosts for AIVs and even highly pathogenic avian influenza (HPAI). Consequently, we sought to identify pigeon RIG-I and investigate its roles in the detection of A/Chicken/Shandong/ZB/2007 (H9N2) (ZB07), Gansu/Tianshui (IBDV TS) and Beijing/CJ/1980 (IBDV CJ-801) strains in chicken DF-1 fibroblasts or human 293T cells. Pigeon mRNA encoding the putative pigeon RIG-I analogs was identified. The exogenous expression of enhanced green fluorescence protein (EGFP)-tagged pigeon RIG-I and caspase activation and recruitment domains (CARDs), strongly induced antiviral gene (IFN-β, Mx, and PKR) mRNA synthesis, decreased viral gene (M gene and VP2) mRNA expression, and reduced the viral titers of ZB07 and IBDV TS/CJ-801 virus strains in chicken DF-1 cells, but not in 293T cells. We also compared the antiviral abilities of RIG-I proteins from waterfowl (duck and goose) and pigeon. Our data indicated that waterfowl RIG-I are more effective in the induction of antiviral genes and the repression of ZB07 and IBDV TS/CJ-801 strain replication than pigeon RIG-I. Furthermore, chicken melanoma differentiation associated gene 5(MDA5)/ mitochondrial antiviral signaling (MAVS) silencing combined with RIG-I transfection suggested that pigeon RIG-I can restore the antiviral response in MDA5-silenced DF-1 cells but not in MAVS-silenced DF-1 cells. In conclusion, these results demonstrated that pigeon RIG-I and CARDs have a strong antiviral ability against AIV H9N2 and IBDV in chicken DF-1 cells but not in human 293T cells. Full article
(This article belongs to the Section Animal Viruses)
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Figure 1

Figure 1
<p>Amino acid alignment of pigeon, goose, duck and zebra finch RIG-I. Black shading indicates amino acid identity, and the green frame indicates the phosphorylation sites. The blue frame denotes ubiquitination sites, and the red frame indicates the ATP-binding motif. The yellow frame indicates the critical residue for the interaction between CARDs and HEL2i, and the orange frame denotes the critical residues for RNA ligand binding.</p> Full article ">Figure 2
<p>Overexpression of pigeon RIG-I in chicken DF-1 cells and human 293T cells (<b>A</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP. Then, 24 h later, fluorescence microscopy was utilized to examine the EGFP, pCARDs-EGFP and pRIG-I-EGFP expression; (<b>B</b>) 293T cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP. Then, 24 h later, fluorescence microscopy was utilized to examine the EGFP, pCARDs-EGFP and pRIG-I-EGFP expression; (<b>C</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, and 24 h later cell lysates were separated using SDS-PAGE and probed with anti-EGFP; (<b>D</b>) 293T cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, and 24 h later cell lysates were separated using SDS-PAGE and probed with anti-EGFP.</p> Full article ">Figure 3
<p>Localization of pigeon CARDs and RIG-I in chicken DF-1 cells and human 293T cells (<b>A</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP (ppigeonCARDs-EGFP) and ppRIG-I-EGFP (ppigeonRIG-I-EGFP), and 24 h later, transfected cells were dyed with DAPI, then, the cellular localization of pRIG-I and CARDs was examined via confocal microscopy; (<b>B</b>) 293T cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, and 24 h later, the transfected cells were dyed with DAPI, then, the cellular localization of pRIG-I and CARDs was examined via confocal microscopy.</p> Full article ">Figure 4
<p>Co-localization of pigeon CARDs with MAVS (<b>A</b>) phMAVS-flag and ppRIG-I-EGFP/ppCARDs-EGFP were co-transfected into 293T cells respectively, and 24 h later, transfected cells were infected with ZB07 viruses or mock-treated, then, transfected cells were dyed with anti-flag-antibody and DAPI at 8 h p.i. Then, the cellular localization of pRIG-I, CARDs, and hMAVS was examined via confocal microscopy; (<b>B</b>) pcMAVS-flag and ppRIG-I-EGFP were co-transfected into DF-1 cells respectively, and 24 h later, transfected cells were infected with ZB07 viruses or mock-treated, then, transfected cells were dyed with anti-flag antibody and DAPI at 8 h p.i. Then, the cellular localization of pRIG-I and hMAVS was examined via confocal microscopy; (<b>C</b>) 293T cells were co-transfected with ppCARDs-EGFP and ppCARDs-flag. 24 h later, transfected cells were dyed with DAPI, anti-flag antibody, and cy3 labled secondary antibody. Then, the cellular localization of pCARDs-EGFP and CARDs-flag was examined via confocal microscopy.</p> Full article ">Figure 5
<p>IBDV and ZB07 induced antiviral gene expression in pigeon CARDs and RIG-I transfected DF-1 and 293T cells. (<b>A–C</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, then, 24 h later, the transfected cells were infected with CJ-801, TS, ZB07 viruses or mock-treated. RNA was extracted and the <span class="html-italic">IFN-</span>β, <span class="html-italic">Mx</span>, and <span class="html-italic">PKR</span> mRNA expression were determined by qRT-PCR at 8 h p.i.; (<b>D</b>) 293T cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP. Then, 24 h later, the transfected cells were infected with CJ-801, TS, ZB07 viruses or mock-treated. RNA was extracted and the <span class="html-italic">IFN-</span>β mRNA expression were determined by qRT-PCR at 8 h p.i.; (<b>E</b>) DF-1 cells were transfected with ppCARDs-EGFP, ppRIG-I-EGFP, and pEGFP-N1, then, 24 and 48 h later, metabolic activity was measured using the MTT assay; (<b>F</b>) DF-1 cells were transfected with ppCARDs-EGFP, ppRIG-I-EGFP, and pEGFP-N1. Then, 24 and 48 h later, crystal violet staining was used to assess cytotoxic effects. Data represent mean ± SEM from three wells per group. Results are representative of three independent experiments. Data represent mean ± SEM from three wells per group. <b>*</b> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1; <b>**</b> <span class="html-italic">p</span> ≤ 0.01 <span class="html-italic">vs.</span> pEGFP-N1; <b>***</b> <span class="html-italic">p</span> ≤ 0.001 <span class="html-italic">vs.</span> pEGFP-N1. Results are representative of two independent experiments.</p> Full article ">Figure 5 Cont.
<p>IBDV and ZB07 induced antiviral gene expression in pigeon CARDs and RIG-I transfected DF-1 and 293T cells. (<b>A–C</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, then, 24 h later, the transfected cells were infected with CJ-801, TS, ZB07 viruses or mock-treated. RNA was extracted and the <span class="html-italic">IFN-</span>β, <span class="html-italic">Mx</span>, and <span class="html-italic">PKR</span> mRNA expression were determined by qRT-PCR at 8 h p.i.; (<b>D</b>) 293T cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP. Then, 24 h later, the transfected cells were infected with CJ-801, TS, ZB07 viruses or mock-treated. RNA was extracted and the <span class="html-italic">IFN-</span>β mRNA expression were determined by qRT-PCR at 8 h p.i.; (<b>E</b>) DF-1 cells were transfected with ppCARDs-EGFP, ppRIG-I-EGFP, and pEGFP-N1, then, 24 and 48 h later, metabolic activity was measured using the MTT assay; (<b>F</b>) DF-1 cells were transfected with ppCARDs-EGFP, ppRIG-I-EGFP, and pEGFP-N1. Then, 24 and 48 h later, crystal violet staining was used to assess cytotoxic effects. Data represent mean ± SEM from three wells per group. Results are representative of three independent experiments. Data represent mean ± SEM from three wells per group. <b>*</b> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1; <b>**</b> <span class="html-italic">p</span> ≤ 0.01 <span class="html-italic">vs.</span> pEGFP-N1; <b>***</b> <span class="html-italic">p</span> ≤ 0.001 <span class="html-italic">vs.</span> pEGFP-N1. Results are representative of two independent experiments.</p> Full article ">Figure 6
<p>IBDV and ZB07 replication in pigeon CARDs and RIG-I transfected DF-1 cells. (<b>A</b>–<b>C</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, and 24 h later, the transfected cells were infected with CJ-801, TS, and ZB07 viruses (MOI = 0.01). At 24 and 48 h p.i., the viral titers in the cell cultures were determined using TCID50. (<b>D</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP and ppRIG-I-EGFP, and 24 h later, the transfected cells were infected with TS and ZB07 viruses. The RNA was extracted from cells for qRT-PCR at 8 h p.i. Data represent mean ± SEM from three wells per group. <b>*</b> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1; <b>**</b> <span class="html-italic">p</span> ≤ 0.01 <span class="html-italic">vs.</span> pEGFP-N1; <b>***</b> <span class="html-italic">p</span> ≤ 0.001 <span class="html-italic">vs.</span> pEGFP-N1. Results are representative of two independent experiments.</p> Full article ">Figure 7
<p>Comparison between waterfowl and pigeon RIG-I in their abilities to induce antiviral response against IBDV and ZB07 in DF-1 cells. (<b>A</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP, pdCARDs-EGFP (pduckCARDs-EGFP), pgCARDs-EGFP (pgooseCARDs-EGFP), and 24 h later, the transfected cells were infected with CJ-801, TS, and ZB07 viruses or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i.; (<b>B</b>) DF-1 cells were transfected with ppRIG-I-EGFP, pdRIG-I-EGFP (pduckRIG-I-EGFP), pgRIG-I-EGFP (pgooseRIG-I-EGFP), and pEGFP-N1. Then, 24 h later, the transfected cells were infected with CJ-801, TS and ZB07 viruses or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i. Data represent mean ± SEM from three wells per group. <sup>α</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1, <sup>β</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> ppCARDs-EGFP, <sup>γ</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pdCARDs-EGFP, <sup>a</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1, <sup>b</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> ppRIG-I-EGFP, <sup>c</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pdRIG-I-EGFP. Results are representative of two independent experiments.</p> Full article ">Figure 7 Cont.
<p>Comparison between waterfowl and pigeon RIG-I in their abilities to induce antiviral response against IBDV and ZB07 in DF-1 cells. (<b>A</b>) DF-1 cells were transfected with pEGFP-N1, ppCARDs-EGFP, pdCARDs-EGFP (pduckCARDs-EGFP), pgCARDs-EGFP (pgooseCARDs-EGFP), and 24 h later, the transfected cells were infected with CJ-801, TS, and ZB07 viruses or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i.; (<b>B</b>) DF-1 cells were transfected with ppRIG-I-EGFP, pdRIG-I-EGFP (pduckRIG-I-EGFP), pgRIG-I-EGFP (pgooseRIG-I-EGFP), and pEGFP-N1. Then, 24 h later, the transfected cells were infected with CJ-801, TS and ZB07 viruses or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i. Data represent mean ± SEM from three wells per group. <sup>α</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1, <sup>β</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> ppCARDs-EGFP, <sup>γ</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pdCARDs-EGFP, <sup>a</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pEGFP-N1, <sup>b</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> ppRIG-I-EGFP, <sup>c</sup> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> pdRIG-I-EGFP. Results are representative of two independent experiments.</p> Full article ">Figure 8
<p>Effect of pRIG-I transfection on IFN synthesis in MDA5-knockdown DF-1 cells. (<b>A</b>) siRNA targeting chicken MDA5 and negative control siRNA were transfected into DF-1 cells. Then, 36 h later, the interference efficiency was measured by qRT-PCR; (<b>B</b>,<b>C</b>) DF-1 cells were co-transfected with pigeon RIG-I/pEGFP-N1 and MDA-siRNA/control siRNA (CTR-siRNA), and 36 h later, the transfected cells were infected with IBDV and ZB07 or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i. Data represent mean ± SEM from three wells per group. <b>*</b> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> CTR-siRNA; <b>**</b> <span class="html-italic">p</span> ≤ 0.01 <span class="html-italic">vs.</span> CTR-siRNA; <b>***</b> <span class="html-italic">p</span> ≤ 0.001 <span class="html-italic">vs.</span> CTR-siRNA. Results are representative of two independent experiments.</p> Full article ">Figure 9
<p>Effect of pRIG-I transfection on antiviral response in MAVS-knockdown DF-1 cells. (<b>A</b>) siRNA targeting chicken MAVS and negative control siRNA were transfected into DF-1 cells. Then, 36 h later, the interference efficiency was measured by qRT-PCR; (<b>B</b>,<b>C</b>) DF-1 cells were co-transfected with ppRIG-I-EGFP/pEGFP-N1 and MAVS-siRNA/control siRNA (CTR-siRNA), 36 h later, the transfected cells were infected with IBDV and ZB07 or mock-treated (MOI = 1). qRT-PCR was performed on extracted RNA at 8 h p.i. Data represent mean ± SEM from three wells per group. <b>*</b> <span class="html-italic">p</span> ≤ 0.05 <span class="html-italic">vs.</span> CTR-siRNA; <b>**</b> <span class="html-italic">p</span> ≤ 0.01 <span class="html-italic">vs.</span> CTR-siRNA; <b>***</b> <span class="html-italic">p</span> ≤ 0.001 <span class="html-italic">vs.</span> CTR-siRNA. Results are representative of two independent experiments.</p> Full article ">
618 KiB  
Article
Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes
by Yu-Fu Hung, Melanie Schwarten, Silke Hoffmann, Dieter Willbold, Ella H. Sklan and Bernd W. Koenig
Viruses 2015, 7(7), 4119-4130; https://doi.org/10.3390/v7072812 - 21 Jul 2015
Cited by 25 | Viewed by 6743
Abstract
Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell [...] Read more.
Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1–48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A. Full article
(This article belongs to the Special Issue Viral Replication Complexes: Structures, Functions, Applications and Inhibitors)
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Figure 1

Figure 1
<p>Liposome floatation assay of wild type and mutant NS4A(1–48). Alexa-488-labeled NS4A(1–48) (<b>A</b>); Alexa-488-labeled NS4A(1–48, L6E;M10E) (<b>B</b>); or free Alexa Fluor 488 dye (<b>C</b>) were mixed with sonicated POPC liposomes and loaded with the 35% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) sucrose layer of a sucrose step gradient schematically shown on the left; Alexa-488-labeled NS4A(1–48) without liposomes was loaded with the 35% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) sucrose layer in lane (<b>D</b>). Note: The narrow green line at the top of tubes (<b>B</b>), (<b>C</b>) and (<b>D</b>) results from reflection of fluorescence light at the air buffer interface rather than the presence of dye. Fluorescence images of the four tubes recorded prior to ultracentrifugation are shown in the lower row.</p> Full article ">Figure 2
<p>Intensity of backbone amide cross peaks in HSQC spectra of NS4A(1–48) (<b>A</b>) and NS4A(1–48, L6E;M10E) (<b>B</b>) recorded at various lipid concentrations. Peak intensities measured at 2.5 (green), 5 (red) and 10 mg·mL<sup>−1</sup> POPC (black) in the sample were normalized to the intensity of the same signal observed in lipid-free buffer and are shown as a function of the amino acid sequence of the studied peptide. Cross peaks not observed in the lipid-free sample are indicated by minus signs. Cross peaks that are present in buffer but completely disappear after addition of 2.5 mg·mL<sup>−1</sup> POPC are indicated by asterisk.</p> Full article ">
473 KiB  
Review
Exosomes: Implications in HIV-1 Pathogenesis
by Marisa N. Madison and Chioma M. Okeoma
Viruses 2015, 7(7), 4093-4118; https://doi.org/10.3390/v7072810 - 20 Jul 2015
Cited by 140 | Viewed by 15316
Abstract
Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by [...] Read more.
Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission. Full article
(This article belongs to the Special Issue Viruses and Exosomes)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Cell culture derived exosomes modulate HIV-1 infection: (<b>1</b>) Cell-free HIV-1 virions bind host cells via viral envelope (Env) to (<b>2</b>) CD4 on target cell plasma membrane followed by binding to a viral co-receptor, CXCR4 or CCR5; (<b>3</b>) Co-receptor binding initiates viral fusion, whereupon viral contents including (<b>4</b>) single stranded viral genomic RNA (gRNA); reverse transcriptase (RT) and integrase (IN] are emptied into the cellular cytoplasm. Viral gRNA is (<b>5</b>) reverse transcribed by HIV-1 reverse transcriptase (RT) into (<b>6</b>) double stranded viral copy DNA (HIV-1 DNA), which is incorporated by (<b>7</b>) viral IN enzyme as (<b>8</b>) proviral DNA within human genomic DNA (gDNA). The provirus is transcribed by host cellular machinery into (<b>9</b>) nascent viral RNA, which is translated by host cellular machinery into (<b>10</b>) HIV-1 proteins, including Nef. Viral proteins are assembled with nascent viral RNA including HIV TAR RNA sequences (vmiRTAR) into (<b>11</b>) budding progeny virions. During the budding process viral protease initiates cleavage of viral proteins in budding progeny. Budding progeny break free from the cell as (<b>12</b>) immature progeny within the extracellular milieu, which undergo maturation with the completion of viral protease-mediated processing into (<b>13</b>) mature progeny capable of propagating productive HIV-1 infection in neighboring and distal cells; (<b>14</b>) Inward budding of the cellular plasma membrane during endocytosis generates endosomes; (<b>15</b>) Inward budding of the endosome membrane generates exosomes with cellular cytosol derived proteins and nucleotides sequestered within the exosome lumen, including HIV Nef and vmiRTAR; (<b>16</b>) Back fusion of the endosome containing exosomes (multivesicular body) to the cellular plasma membrane releases exosomes into the extracellular milieu. Exosomes derived from HIV-1 infected cell cultures may enhance infection through the following mechanisms: (<b>17</b>) transferring viral binding and entry receptors to HIV-1 susceptible cells, thereby increasing the expression of viral binding sites on the surface of the host cellular plasma membrane. (<b>18</b>) Nef-mediated internalization and degradation of CD4 molecules and Nef-mediated reduction of CD4 expression on the surface of exosomes and (<b>19</b>) suppression of Bim and Cdk9 expression and apoptosis by viral miRNA generated from vmiRTAR. The protective roles of cell culture derived exosomes from uninfected cells may be mediated by: (<b>20</b>) Apobec3G enwrapped within exosomes and (<b>21</b>) exosomal CD4 binding to HIV-1 Env.</p> Full article ">Figure 2
<p>Model of biofluid exosome antiviral functions within the HIV-1 life cycle: (<b>1</b>–<b>13</b>) HIV-1 life cycle as defined in <a href="#viruses-07-02810-f001" class="html-fig">Figure 1</a> legend. Biofluid exosomes may interact with free HIV-1 and (<b>14</b>) sequester free viral particles within exosome aggregates thereby inhibiting HIV-1 infection by preventing virus from binding to target cells. Biofluid exosomes that present CD4 molecules on the exosome surface may (<b>15</b>) compete for viral Env binding to CD4 on the host cell plasma membrane, thereby inhibiting HIV-1 infection by preventing virus from binding to target cells. Competition may also occur via binding of exosome ligands/receptors to other receptors/ligands on the viral envelope (e.g., phosphatidylserine/annexin interaction). Exosomes are taken up by cells via direct fusion with the plasma membrane or by endocytosis into endosomes that may subsequently fuse with lysosomes. Exosomal interaction with HIV-1 may lead to entry of the virus into the cell via endocytosis, leading to (<b>16</b>) exosomal delivery of virus into lysosomes for degradation, subsequently inhibiting HIV-1 infection. (<b>17</b>) HIV-1 gRNA may be degraded or otherwise rendered non-functional by exosomal antiviral proteins including Apobec3g, or following translation of exosomal antiviral mRNA into antiviral proteins including Apobec3g, or by antiviral exosomal miRNA. Exosomal antiviral protein, mRNA or miRNA may (<b>18</b>) inhibit viral RT and reverse transcription processes by blocking RT activity, blocking RT binding to RNA or facilitating degradation of RT. In the event that exosomes fuse with the cellular plasma membrane, they may enrich the cell surface with proteins that may function to (<b>19</b>) tether budding progeny virions to the plasma membrane (e.g., BST-2/tetherin, PS/Annexin), preventing them from diffusing into the extracellular milieu and subsequently preventing HIV-1 propagation.</p> Full article ">Figure 3
<p>Biofluid derived exosomes modulate HIV-1 infection: (<b>1</b>–<b>6</b>) HIV-1 life cycle as defined in <a href="#viruses-07-02810-f001" class="html-fig">Figure 1</a> legend. Exosomes derived from human semen inhibit direct, <span class="html-italic">trans</span> and cell-to-cell transmission of HIV in human PBL, T cells and monocytes by (<b>7</b>) mediating deleterious effects on HIV reverse transcriptase and reverse transcription processes. Exosomes derived from human blood plasma or blood serum have no effect on HIV infectivity. Exosomes derived from human breast milk inhibit cell-to-cell transmission of HIV from monocyte-derived dendritic cells (MDDC) to CD4+ T cells by (<b>8</b>) competing for binding of HIV to DC-SIGN on MDDC. Biofluid derived exosomes may also inhibit HIV infectivity by (<b>9</b>) exosomal donation or transfer of antiviral cargo to recipient cells; (<b>10</b>) inhibition of cellular signaling or molecules required for HIV replication or (<b>11</b>) induction of cellular signaling or donation of exosomal factors resulting in enhancement of expression of molecules responsible for host protection against HIV.</p> Full article ">
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Article
Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy
by Marion Adelfinger, Simon Bessler, Alexa Frentzen, Alexander Cecil, Johanna Langbein-Laugwitz, Ivaylo Gentschev and Aladar A. Szalay
Viruses 2015, 7(7), 4075-4092; https://doi.org/10.3390/v7072811 - 20 Jul 2015
Cited by 31 | Viewed by 8748
Abstract
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody [...] Read more.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS. Full article
(This article belongs to the Special Issue Oncolytic Viruses)
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<p>VEGF expression in MTH52c, ZMTH3, CT1258 or STSA-1 canine cancer cells under cell culture conditions. Each value represents the mean (<span class="html-italic">n</span> = 3) +/− standard deviations (SD).</p> Full article ">Figure 2
<p>Relative survival of MTH52c, ZMTH3, CT1258 or STSA-1 canine cancer cells after LIVP 6.1.1 or GLV-5b451 infection at an MOI of 0.1. Viable cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Mean values (<span class="html-italic">n</span> = 3) and standard deviations are shown as percentages of respective controls. The data represent two independent experiments.</p> Full article ">Figure 3
<p>Replication capacity of the vaccinia virus strains LIVP 6.1.1 (<b>A</b>) and GLV-5b451 (<b>B</b>) in different canine cancer cell lines. For the viral replication assay, MTH52c, ZMTH3, CT1258 or STSA-1 cells grown in 24-well plates were infected with either LIVP 6.1.1 or GLV-5b451 at an MOI of 0.1. Cells and supernatants were collected for the determination of virus titers at various time points. Viral titers were determined as pfu per ml in triplicates by standard plaque assay in CV-1 cell monolayers. Averages plus standard deviation are plotted. The data represent three independent experiments.</p> Full article ">Figure 4
<p>Expression of GLAF-2 protein in different canine cancer cells: (<b>A</b>) MTH52c; <b>(B</b>) ZMTH3; (<b>C</b>) CT1258 and (<b>D</b>) STSA-1. Western blot analysis of GLV-5b451-infected (MOI of 1.0; lines marked by *) or uninfected canine cancer cells. Protein fractions from cell lysates were isolated at 1, 24, 48, 72 and 96 h post virus infection and separated by SDS-PAGE. Western blot analysis was performed as described in material and methods. The position of GLAF-2 protein is marked by black arrow. M: PageRuler Prestained Protein Ladder # 26616 (Thermo Scientific, Bonn, Germany). The positions of the 35 and 25 kDa proteins are marked by $ symbol.</p> Full article ">Figure 5
<p>Interactions of purified GLAF-2 antibodies with canine, murine, and human VEGFs. Affinity and cross reactivity of GLAF-2 was demonstrated by ELISA. Equal concentrations of canine, murine, or human VEGF (100 ng/well) were coated on ELISA plates. Seven two-fold dilutions of purified GLAF-2 proteins ranging from 2000 ng/mL to 31.3 ng/mL were incubated with canine, murine and human VEGFs. PBS was used as negative control. For further ELISA experimental conditions see material and methods. ODs obtained for various concentrations of GLAF-2 against canine, murine and human VEGF were plotted. ELISA was repeated in three independent experiments. Each value represents the mean (<span class="html-italic">n</span> = 3) +/− standard deviations (SD).</p> Full article ">Figure 6
<p>Effects on tumor growth (<b>A</b>) and body weights (<b>B</b>) of virus- and mock-treated STSA-1 xenografted mice. (A) Groups of STSA-1-tumor-bearing nude mice (<span class="html-italic">n</span> = 7) were either treated with a single dose of 1 × 10<sup>7</sup> pfu GLV-5b451 or LIVP 6.1.1 or with PBS (mock control). Statistical analysis was performed with a paired Student’s <span class="html-italic">t</span>-test (* <span class="html-italic">p</span> &lt; 0.05); (B) Relative mean weight changes of STSA-1 cell xenografted mice after virus or PBS treatment. The data are presented as mean values +/− SD.</p> Full article ">Figure 7
<p>Presence of the scAb GLAF-2 (<b>A</b>) and analysis of vascular density in tumor tissues of virus- or PBS-injected STSA-1 xenograft mice at 17 dpvi (<b>B</b>–<b>D</b>). (A) Western blot analysis of GLV-5b451-infected STSA-1 tumors at 17 dpvi (lines 2–4). Line 1: Lysate of a LIVP 6.1.1-infected STSA-1 tumor (negative control). Each sample represents an equivalent of 1.5 mg tumor mass; (B,C) Visualization and analysis of vascular density using CD31 immunohistochemistry in LIVP 6.1.1, GLV-5b451 or PBS-treated tumors: (B) The vascular density was measured in CD31-labeled tumor cross-sections (<span class="html-italic">n</span> = 3 mice per group, 24 images per virus-injected mouse or 12 images per control PBS mouse) and presented as mean values +/− SD. (*** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01;* <span class="html-italic">p</span> &lt; 0.05 Student’s <span class="html-italic">t</span>-test); (<b>C</b>) Fluorescence intensity of the CD31 signal of blood vessels. The fluorescence intensity of the CD31-labelling represents the average brightness of all vessel-related pixels and was determined as described in materials and methods. Shown are the mean values +/− standard deviations. Statistical analysis was performed with a two-tailed unpaired Student’s <span class="html-italic">t</span>-test (*** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05); (D) Representative tumor sections labeled with anti-CD31 antibody (red) and anti-vaccinia virus (VV) antibody (green). Scale bars: 150 mm.</p> Full article ">
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Review
The Emerging Role of miRNAs in HTLV-1 Infection and ATLL Pathogenesis
by Ramona Moles and Christophe Nicot
Viruses 2015, 7(7), 4047-4074; https://doi.org/10.3390/v7072805 - 20 Jul 2015
Cited by 28 | Viewed by 8807
Abstract
Human T-cell leukemia virus (HTLV)-1 is a human retrovirus and the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a fatal malignancy of CD4/CD25+ T lymphocytes. In recent years, cellular as well as virus-encoded microRNA (miRNA) have been shown to deregulate signaling pathways to [...] Read more.
Human T-cell leukemia virus (HTLV)-1 is a human retrovirus and the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a fatal malignancy of CD4/CD25+ T lymphocytes. In recent years, cellular as well as virus-encoded microRNA (miRNA) have been shown to deregulate signaling pathways to favor virus life cycle. HTLV-1 does not encode miRNA, but several studies have demonstrated that cellular miRNA expression is affected in infected cells. Distinct mechanisms such as transcriptional, epigenetic or interference with miRNA processing machinery have been involved. This article reviews the current knowledge of the role of cellular microRNAs in virus infection, replication, immune escape and pathogenesis of HTLV-1. Full article
(This article belongs to the Special Issue Recent Advances in HTLV Research 2015)
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<p>Human T-cell leukemia virus HTLV-1 interferes with cellular miRNA machinery. MiRNAs are transcribed by the RNA polymerase II or III into the nucleus as primary miRNAs (pri-miRNAs) from coding or non-coding part of genes. The nuclear RNase III Drosha recognized and processed pri-miRNAs into a hairpin-shaped RNA, named precursor miRNAs. Pre-miRNAs are transported to the cytoplasm by Exportin 5, and processed by the cytoplasmic RNase III Dicer in the mature miRNA duplex. The duplex forms a complex named RNA-Induced Silencing Complex (RISC). MiRNAs bind complementary sequences usually localized at 3′UTR of messenger RNA and this binding results in the inhibition of translation and/or messenger RNA degradation. HTLV-1 deregulates the cellular miRNA pathway by suppressing the function of Drosha and Dicer. Tax directly interacts with Drosha and the binding leads to Drosha degradation mediated by proteasome complex. The regulatory protein, Rex, is reported to directly interact with Dicer. Rex suppresses the ribonuclease-directed processing activity of Dicer, protecting against the cleavage Rex-mRNA.</p> Full article ">Figure 2
<p>MiR-28-3p targets the HTLV-1 genome. The figure illustrates a natural feedback loop that regulated cellular miRNA expression in response to virus infection. MiR-28-3p suppresses HTLV-1 expression by targeting a sequence localized within the viral gag/pol HTLV-1 sequence. MiR-28-3p expression leads to abortive infection by inhibiting HTLV-1 reverse transcription and preventing the formation of the pre-integration complex.</p> Full article ">Figure 3
<p>MiRNAs promote cell proliferation. MiR-155 and miR-146a were found elevated in HTLV-1-infected cells <span class="html-italic">in vitro.</span> Tax induces the transcription factors NF-κB and AP-1, which promote miR-155 expression by binding the miRNA promoter. This binding resulted in an increased expression of the B-cell integration cluster (BIC) gene whose transcript is processed into miR-155. The interferon regulatory factor-4, IRF4, which is induced in HTLV-1-infected cells, promotes BIC/miR-155 expression. NF-κB also mediates miR-146a transactivation; both miRNAs enhance cellular growth in HTLV-1-infected cells. MiR-150 and miR-223 are differentially regulated in ATLL samples and in HTLV-1-transformed cells. MiR-150 and miR-223 were found upregulated in acute ATLL patients and downregulated in HTLV-1-transformed cell lines. MiR-150 and miR-223 target the STAT1 3′UTR. Inhibition of STAT1 expression, through miR-150, miR-223 reduced proliferation of HTLV-1-transformed and ATLL-derived cell lines. MiR-150 and miR-223, by decreasing STAT1 expression and dampening STAT1-dependent signaling in human T cells, regulated proliferation in an HTLV-1 context.</p> Full article ">Figure 4
<p>MiRNAs induce resistance to apoptosis. MiR-31 is one of the most profoundly repressed miRNAs in primary ATLL cells. The Polycomb protein complex is overexpressed in ATLL cells and suppresses miR-31 expression. MiR-31 negatively regulates NF-κB-inducing kinase (NIK) and leads to apoptosis resistance. MiR-130b and miR-93 are upregulated in HTLV-1 cell lines and ATLL patients and both target Tumor protein p53-inducible nuclear protein (TP53INP1). TP53INP1 is a tumor suppressor gene that has anti-proliferative and pro-apoptotic activities via both p53-dependent and p53-independent means. TP53INP1 has in its 3′ UTR two binding sites for miR-93 and two sites for miR-130b.</p> Full article ">Figure 5
<p>MiR-149 and miR-873 promote chromatin remodeling. The Tax protein promotes HTLV-1 gene expression by its interaction with the long terminal repeat (LTR) or U3 region of the viral promoter. To activate the transcription, Tax recruits the p300/CREB-binding protein (p300/CBP) and p300/CBP-associated factor (P/CAF), which bind two different regions of Tax, resulting in histone acetylation and chromatin remodeling. MiR-149 and miR-873 are downregulated in HTLV-1-transformed cell lines and target the chromatin remodeling factors p300 and p/CAF.</p> Full article ">Figure 6
<p>MiRNAs induce genetic instability. MiR-17 and miR-21 are upregulated in an HTLV-1 context. HBZ inactivates OBFC2A via miR-17 and miR-21, promoting genetic instability and cell proliferation. OBFC2A encodes for hSSB2, which is involved in the ATM signaling pathway, the activation of the cell cycle checkpoint and promotes DNA repair.</p> Full article ">
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Review
Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design
by Alexander W. Tarr, Tanvi Khera, Kathrin Hueging, Julie Sheldon, Eike Steinmann, Thomas Pietschmann and Richard J. P. Brown
Viruses 2015, 7(7), 3995-4046; https://doi.org/10.3390/v7072809 - 17 Jul 2015
Cited by 39 | Viewed by 10166
Abstract
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have [...] Read more.
In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design. Full article
(This article belongs to the Special Issue Viral Glycoprotein Structure)
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<p>Comparison of the structures of the HCV E2 glycoprotein core [<a href="#B42-viruses-07-02809" class="html-bibr">42</a>] with the crystal structures of Bovine Viral Diarrhoea Virus E2 glycoprotein [<a href="#B48-viruses-07-02809" class="html-bibr">48</a>] and Tick-Borne Encephalitis virus E glycoprotein [<a href="#B67-viruses-07-02809" class="html-bibr">67</a>]. The resolved crystal structure of HCV E2 revealed a much more compact protein structure than that of other members of the family Flaviviridae. Protein ribbons are coloured from the N-terminus (blue) through to the C-terminus (red). The PDB ID numbers are indicated in brackets.</p> Full article ">Figure 2
<p>Structural flexibility in the N-terminus of E2. Peptides representing the amino acids 412–424 of the HCV polyprotein (blue) were crystalized in complex with monoclonal antibodies (in each case the heavy chain is highlighted in white and light chain in grey). (<b>A</b>) AP33 [<a href="#B63-viruses-07-02809" class="html-bibr">63</a>,<a href="#B65-viruses-07-02809" class="html-bibr">65</a>]; (<b>B</b>) HC33.1 [<a href="#B66-viruses-07-02809" class="html-bibr">66</a>]; (<b>C</b>) HCV1 [<a href="#B65-viruses-07-02809" class="html-bibr">65</a>]; (<b>D</b>) 3/11 [<a href="#B62-viruses-07-02809" class="html-bibr">62</a>]. The contrasting structures of this region when in complex with different antibodies, ranging from a β-turn structure (<b>A</b>,<b>C</b>) to an extended coil (<b>B</b>,<b>D</b>). This suggests that the conserved N-terminus of E2 can adopt an array of alternative conformations on the surface of the protein. This inherent flexibility could be a mechanism to evade neutralizing antibody responses, or could highlight the different conformations adopted at different stages of the entry cascade.</p> Full article ">Figure 3
<p>Characterization of positive and negative selection in HCV subtype 1a and 1b envelope glycoproteins. For each panel, full-length E1E2 nucleotide sequences were downloaded from GenBank and aligned according to encoded amino acid sequence. A comparative analysis of selection acting on contemporaneously sampled subtype 1a (<b>A</b>) and 1b (<b>B</b>) glycoprotein sequences is presented. For both data sets, normalized dN-dS values across the E1E2 coding-region are presented, with the test statistic obtained using the total number of substitutions in the tree (measured in expected substitutions per site). The test statistic dN-dS is used for detecting codons that have undergone positive selection, where dS is the number of synonymous substitutions per site (s/S) and dN is the number of nonsynonymous substitutions per site (n/N). Positive values indicate an overabundance of non-synonymous substitutions and are indicative of adaptive evolution. Negative values are indicate an overabundance of synonymous substitutions and are indicative of purifying selection due to structural/functional constraint. The numbers of sequences analysed for each genotype are presented in the top right corner of each plot. A magnified plot of selection in HVR1 (E2 residues 384–410) is positioned below each full-length E1E2 selection plot, with positively selected sites highlighted in red and negatively selected sites highlighted in blue. Subtype 1a and subtype 1b HVR1 consensus amino acids are located below each HVR1 plot. Phylogenetic trees representing the sequences utilized for each analysis are positioned to the right of each E1E2/HVR1 plot. Branch lengths are equivalent to genetic distance measured in nucleotide substitutions per site and are proportional to the scale bar. Maximum Likelihood computations of dN and dS were conducted using HyPhy [<a href="#B97-viruses-07-02809" class="html-bibr">97</a>] and MEGA6 [<a href="#B98-viruses-07-02809" class="html-bibr">98</a>].</p> Full article ">Figure 4
<p>Conservation and variability in the E1 and E2 proteins. Conservation plots were generated using an alignment of 866 genotype 1 full-length E1E2 amino acid sequences retrieved from GenBank. Alignments were plotted using the ConSurf server [<a href="#B99-viruses-07-02809" class="html-bibr">99</a>], highlighting the variability in the first 79 amino acids of E1 (mapped onto pdb 4UOI), and the core structure of E2 (mapped onto pdb 4MWF). Conserved amino acids are represented in mauve, and highly variable amino acids in cyan. This analysis revealed a mainly conserved exposed face of the N-terminus of E1, with some variable residues buried in the monomer of the N-terminus of E1. It also showed two conserved faces of the core E2 structure (highlighted by contiguous mauve residues in the two diagrams), with a variable discontinuous region (highlighted with patches of cyan residues) surrounding the conserved CD81 binding residues and neutralizing epitopes.</p> Full article ">Figure 5
<p>Cartoon of the protein constructs used for crystallization of E1 (71) and E2 [<a href="#B42-viruses-07-02809" class="html-bibr">42</a>,<a href="#B49-viruses-07-02809" class="html-bibr">49</a>] mapped onto the HCV E1 and E2 genes (blue). Regions possessing antibody epitopes are highlighted in red; glycosylation sites are indicated by a green branching structure; and hypervariable regions are highlighted in yellow. The numbering of the ends of the protein constructs are highlighted. A red dashed line represents a Gly-Ser-Ser-Gly linker added between two regions in the E2 core construct reported by Kong <span class="html-italic">et al.</span>, 2013 [<a href="#B42-viruses-07-02809" class="html-bibr">42</a>].</p> Full article ">Figure 6
<p>Proposed structure of the E2 protein, highlighting hyrophobic side chains (red) and hydrophilic side chains (blue) on the conserved E2 core crystal. Unresolved regions including the conserved N-terminal domain, HVR1 and the unresolved region of the ectodomain (aa646–715) are highlighted as blue ovals. The G-S-S-G linker used to replace the HVR2 in the structure presented by Kong et al is identified by a dashed red oval labelled “HVR2”. The transmembrane domain (aa 716–745) is highlighted in yellow. Three separate linear antigenic clusters are highlighted, which make a contiguous antigenic region that possesses many antibody epitopes (reviewed in Edwards <span class="html-italic">et al.</span>, 2012 [<a href="#B152-viruses-07-02809" class="html-bibr">152</a>]). Most of these epitopes are linear and not dependent of the overall conformation of the E2 core. Some antibodies (such as AR3A, AR3B, AR3C, and e137) recognize overlapping conformation-dependent discontinuous epitopes.</p> Full article ">
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Review
Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics
by Ester Sesmero and Ian F. Thorpe
Viruses 2015, 7(7), 3974-3994; https://doi.org/10.3390/v7072808 - 17 Jul 2015
Cited by 44 | Viewed by 9575
Abstract
Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is [...] Read more.
Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process. Full article
(This article belongs to the Special Issue Viral Replication Complexes: Structures, Functions, Applications and Inhibitors)
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<p>(<b>a</b>) Right-hand structure of HCV polymerase (NS5B). Palm, fingers and thumb domains are shown in red, blue and green respectively; (<b>b</b>) Duplex channel in NS5B (front of the enzyme); (<b>c</b>) NTP channel in NS5B (back of the enzyme); (<b>d</b>) Motifs and functional regions of NS5B. Motif A in red, B in orange, C in yellow, D in bright green, E in pink, F in purple and G in cyan. Functional regions: I in light green, II in violet and III in tan; (<b>e</b>) Template channel (top view of the enzyme).</p> Full article ">Figure 2
<p>Schematic describing de novo initiation in Hepaciviruses and Pestiviruses. Note that Flaviruses do not anchor their C-terminus in the Endoplasmatic Reticulum (ER). This figure was generated by incorporating the descriptions provided by both Appleby <span class="html-italic">et al.</span> [<a href="#B35-viruses-07-02808" class="html-bibr">35</a>] and Choi [<a href="#B1-viruses-07-02808" class="html-bibr">1</a>]. The linker and C-terminal anchor are shown in orange as one contiguous element. The β-flap is colored red (as in <a href="#viruses-07-02808-f003" class="html-fig">Figure 3</a>), the template strand in purple, the growing strand in green, the stabilizing GTP in blue and the Endoplasmic Reticulum (ER) in brown. The “N” and “P” indicate where the N-site (nucleotide-site) and P-site (priming-site) are. These correspond to the positions of the growing strand that bind to residues “<span class="html-italic">n</span>” and “<span class="html-italic">n</span> + 1” of the template strand respectively.</p> Full article ">Figure 3
<p>NS5B structure with characteristic elements highlighted. (<b>a</b>) Front view and (<b>b</b>) top view. The linker is shown in orange, the β-flap in red. The fingertips are shown in blue (the delta 1 loop) and green (the delta 2 loop).</p> Full article ">Figure 4
<p>Two metals ions mechanism in RdRps. The squares represent the bases that are part of the nucleotides. This figure is inspired by a similar figure from Choi <span class="html-italic">et al.</span> [<a href="#B1-viruses-07-02808" class="html-bibr">1</a>].</p> Full article ">Figure 5
<p>NS5B inhibitors. (<b>a</b>) The three allosteric sites of NS5B are highlighted with space filling representations of inhibitors that bind in these locations. Thumb site 1 (NNI-1) in yellow, thumb site 2 (NNI-2) in green, palm sites (NNI-3/4) in purple; (<b>b</b>) chemical structures of NIs and NNIs that are in clinical trials or have already been approved [<a href="#B52-viruses-07-02808" class="html-bibr">52</a>,<a href="#B53-viruses-07-02808" class="html-bibr">53</a>,<a href="#B54-viruses-07-02808" class="html-bibr">54</a>,<a href="#B55-viruses-07-02808" class="html-bibr">55</a>,<a href="#B56-viruses-07-02808" class="html-bibr">56</a>,<a href="#B57-viruses-07-02808" class="html-bibr">57</a>,<a href="#B58-viruses-07-02808" class="html-bibr">58</a>,<a href="#B59-viruses-07-02808" class="html-bibr">59</a>].</p> Full article ">Figure 6
<p>Mechanisms of inhibition for NNIs. NS5B must transition between open and closed states to perform replication (upper left). NNI-1 inhibitors have been observed to reduce enzyme stability. NNI-2 inhibitors have been shown to reduce conformational sampling, confining the enzyme in closed conformations. NNI-3 inhibitors mainly block access of the RNA template but also induce some restriction of conformational sampling. The RNA template is represented as a black rectangle and the inhibitor as an orange ellipse.</p> Full article ">
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Article
Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation
by Belén Borrego, Miguel Rodríguez-Pulido, Concepción Revilla, Belén Álvarez, Francisco Sobrino, Javier Domínguez and Margarita Sáiz
Viruses 2015, 7(7), 3954-3973; https://doi.org/10.3390/v7072807 - 17 Jul 2015
Cited by 21 | Viewed by 5805
Abstract
The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and [...] Read more.
The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs). Full article
(This article belongs to the Special Issue Advances in Gene Technology and Resistance to Viruses)
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Figure 1
<p>Induction of IFN-β promoter activity by the FMDV ncRNAs in HEK 293 cells expressing RLRs. (<b>A</b>) Cells (1 × 10<sup>6</sup>) were co-transfected with 50 ng of pIFNβ-FFLuc, 25 ng of pRL-TK, and increasing amounts of flag-RIG-I, flag-MDA5 or empty plasmid (1 and 10 ng). A constitutively active RIG-I, CARD2 (2 ng) served as positive control. After 24 h, cells were mock-treated or stimulated with 0.3 μg (0.2 μg/mL) or 3 μg (2 μg/mL) of the indicated ncRNA. Cells were harvested 24 h later and dual-luciferase assay was performed. The values represent the relative firefly luciferase activity normalized to Renilla luciferase activity and expressed as fold differences relative to mock-transfected cells. Data are representative of two independent experiments and error bars indicate mean +/− SD; (<b>B</b>) Expression of RLRs in transfected 293 cells. HEK293 cells were transfected with 100 ng of flag-RIG-I or flag- MDA5. Forty-eight hours later, cell lysates were prepared and analyzed by immunoblot with anti-flag, anti-RIG-I, anti-MDA5 or anti-β-tubulin antibodies.</p> Full article ">Figure 1 Cont.
<p>Induction of IFN-β promoter activity by the FMDV ncRNAs in HEK 293 cells expressing RLRs. (<b>A</b>) Cells (1 × 10<sup>6</sup>) were co-transfected with 50 ng of pIFNβ-FFLuc, 25 ng of pRL-TK, and increasing amounts of flag-RIG-I, flag-MDA5 or empty plasmid (1 and 10 ng). A constitutively active RIG-I, CARD2 (2 ng) served as positive control. After 24 h, cells were mock-treated or stimulated with 0.3 μg (0.2 μg/mL) or 3 μg (2 μg/mL) of the indicated ncRNA. Cells were harvested 24 h later and dual-luciferase assay was performed. The values represent the relative firefly luciferase activity normalized to Renilla luciferase activity and expressed as fold differences relative to mock-transfected cells. Data are representative of two independent experiments and error bars indicate mean +/− SD; (<b>B</b>) Expression of RLRs in transfected 293 cells. HEK293 cells were transfected with 100 ng of flag-RIG-I or flag- MDA5. Forty-eight hours later, cell lysates were prepared and analyzed by immunoblot with anti-flag, anti-RIG-I, anti-MDA5 or anti-β-tubulin antibodies.</p> Full article ">Figure 2
<p>Mx1 induction in porcine cells transfected with the 3′NCR RNA. SK6 cells (1 × 10<sup>6</sup>) were transfected with 20 μg/mL 3′NCR transcripts. Cells were lysed at 0, 6, 24 or 30 h after transfection. (<b>A</b>) RT-PCR detection of IFN-β, Mx1 and GAPDH mRNAs in RNA extracted from SK6 lysates. Negative controls (water) were included in the RT-PCR assays; (<b>B</b>) Mx1 detection by immunoblot in transfected SK6 cells. Tubulin was used for normalization.</p> Full article ">Figure 3
<p>Analysis of the innate immune response of transfected PBMCs from a single pig (six month-old) over time. (<b>A</b>) Swine PBMCs were transfected with 20 μg/mL IRES or 3′NCR transcripts, with or without Lipofectin, and RNA was extracted at 2, 4, 8 or 24 h pt. Amplification of Mx1 and cyclophilin mRNAs by RT-PCR is shown; (<b>B</b>) Antiviral activity, TNFα, IL-12 and IL-10 levels in supernatants of swine PBMCs (2 × 10<sup>6</sup>) transfected with 3′NCR or IRES as in A or stimulated with poly I:C (10 μg/mL), and collected at 8 or 24 h pt. Mock transfections with culture medium and Lipofectin were performed as negative controls. Antiviral activity is expressed as the reciprocal of the highest dilution of supernatants from transfected PBMCs causing a 50% reduction of the cytophatic effect induced by infection with FMDV on IBRS-2 cells. The levels of the different cytokines were measured by ELISA; (<b>C</b>) Immunoblot detection of Mx1 in lysates of PBMCs transfected with S, IRES, 3′NCR transcripts (20 μg/mL) or mock-transfected using Lipofectin, or stimulated with poly I:C or ODN (both at 10 μg/mL). Cells were lysed 24 h pt. Tubulin was used for normalization.</p> Full article ">Figure 4
<p>Innate responses in transfected PBMCs from different pigs and two independent experiments. (<b>A</b>) PBMCs were isolated from five 9 to 12-month-old pigs and transfected with 20 μg/mL S, IRES, 3′NCR, mock-transfected (negative control), or stimulated with ODN (10 μg/mL) or poly I:C (10 μg/mL) (<span class="html-italic">n</span> = 2). The levels of IFN-α, TNF-α, IL-12 and IL-10 were measured 24 h later by ELISA. Data correspond to individual animals for each group and analysis; (<b>B</b>) PBMCs were isolated from six three-month-old pigs and transfected with IRES, 3′NCR, or mock-transfected as above. Supernatants were tested for antiviral activity (expressed as in <a href="#viruses-07-02807-f003" class="html-fig">Figure 3</a>) at 24 h pt. Individual pig numbers are indicated.</p> Full article ">Figure 5
<p>Effect of ncRNA dosage on cytokine induction in total swine PBMCs or after pDCs-enrichment. (<b>A</b>). PBMCs were transfected with S, IRES or 3′NCR at 5, 20 or 100 μg/mL and the IFN-α, TNF-α, IL10 and IL12 levels were measured by ELISA. The data shown are the ratios of the values obtained at 5 or 100 μg/mL for the indicated cytokine relative to those obtained at 20 μg/mL with the corresponding RNA; (<b>B</b>) Total PBMCs or PBMCs enriched in pDCs by negative selection were transfected with S, IRES or 3′NCR (at 5 or 20 μg/mL) or stimulated with 10 μg/mL ODN. Data indicate levels of IFN-α, TNF-α, IL-10 and IL-12 in pDCs-enriched PBMCs transfected with the ncRNAs relative to those in total PBMCs. IFN-α levels in total or pDCs-enriched PBMCs stimulated with ODN are also shown.</p> Full article ">Figure 6
<p>Effect of BafA1 treatment on cytokine induction in ncRNA-transfected PBMCs. Porcine PBMCs were treated with bafilomycin A1 (250 nM) 15 to 30 min prior to transfection or stimulation. IFN-α, TNF-α and IL-12 levels were measured by ELISA in total PBMCs 24 h after transfection with the ncRNAs (20 μg/mL) or stimulation with ODN (10 μg/mL) or LPS (0.5 μg/mL). Mock-transfections with medium were used as controls. Data are average values plus standard deviations from duplicates of two transfection experiments. Negative samples with OD readings ≤ the mean of blank wells were arbitrarily assigned values of 50, 25, and 100 for IFN-α, TNF-α, and IL-12, respectively, according to the detection limit for each cytokine.</p> Full article ">
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Article
Tsv-N1: A Novel DNA Algal Virus that Infects Tetraselmis striata
by António Pagarete, Théophile Grébert, Olga Stepanova, Ruth-Anne Sandaa and Gunnar Bratbak
Viruses 2015, 7(7), 3937-3953; https://doi.org/10.3390/v7072806 - 17 Jul 2015
Cited by 25 | Viewed by 8430
Abstract
Numbering in excess of 10 million per milliliter of water, it is now undisputed that aquatic viruses are one of the major factors shaping the ecology and evolution of Earth’s microbial world. Nonetheless, environmental viral diversity and roles remain poorly understood. Here we [...] Read more.
Numbering in excess of 10 million per milliliter of water, it is now undisputed that aquatic viruses are one of the major factors shaping the ecology and evolution of Earth’s microbial world. Nonetheless, environmental viral diversity and roles remain poorly understood. Here we report the first thorough characterization of a virus (designated TsV) that infects the coastal marine microalga Tetraselmis striata. Unlike previously known microalgae-infecting viruses, TsV is a small (60 nm) DNA virus, with a 31 kb genome. From a range of eight different strains belonging to the Chlamydomonadaceae family, TsV was only able to infect T. striata. Gene expression dynamics revealed an up-regulation of viral transcripts already 1 h post-infection (p.i.). First clear signs of infection were observed 24 h p.i., with the appearance of viral factories inside the nucleus. TsV assembly was exclusively nuclear. TsV-N1 genome revealed very different from previously known algae viruses (Phycodnaviridae). Putative function and/or homology could be resolved for only 9 of the 33 ORFs encoded. Among those was a surprising DNA polymerase type Delta (only found in Eukaryotes), and two genes with closest homology to genes from human parasites of the urogenital tract. These results support the idea that the diversity of microalgae viruses goes far beyond the Phycodnaviridae family and leave the door open for future studies on implications of microalgae viruses for human health. Full article
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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<p>Host phylogeny and infectivity range of TsV-N1. The tree corresponds to the Maximum Likelihood phylogeny based on 18S sequences of 38 microalgae strains with 1000 bootstrap replicates (bootstrap values indicated above each branch). Bayesian posterior probabilities are indicated below each branch. The susceptible host strain is indicated with a filled symbol, while the other tested/resistant strains have an open symbol. Culture collection reference for the microalgae strains tested for viral infection is indicated between brackets. GenBank accession number is indicated after their name for sequences retrieved from public databases.</p> Full article ">Figure 2
<p>Thin section of a <span class="html-italic">T. striata</span> showing TsV-N1 particles inside (IC) and outside (EC) the cell. Black arrow: filled viral particle, white arrow: empty capsid. Note the hexagonal section of the virions.</p> Full article ">Figure 3
<p>Ultrastructural changes in <span class="html-italic">T. striata</span> associated with the progression of TsV-N1 infection. (<b>A</b>) longitudinal section of an uninfected cell, with anterior flagellar pit and posterior pyrenoid; (<b>B</b>) nucleus of an uninfected cell, view corresponding to the frame in (<b>A</b>); (<b>C</b>) longitudinal section of an infected cell, first noticeable changes; (<b>D</b>) detail of the nucleus corresponding to the frame in (<b>C</b>); first viral capsids, intact nuclear envelope. (<b>E</b>) Longitudinal section of an infected cell with highly disordered nucleus; (<b>F</b>) enlargement of frame in (<b>E</b>) showing a viral factory (vf) inside the nucleus; (<b>G</b>) longitudinal section of a cell at the final stage of infection; (<b>H</b>) enlargement of frame in (<b>G</b>) showing numerous viral particles. Facing black arrowheads (<b>B</b>,<b>D</b>) nuclear envelope. Black and white arrows (<b>D</b>,<b>F</b>,<b>H</b>) full and empty viral particles. Legend: bb, basal bodies; chl, chloroplast; fp, flagellar pit; gol, golgi apparatus; mit, mitochondrion; no, nucleole; nu, nucleus; pyr, pyrenoid; rp, rhizoplast; st, starch grain; and vf, viral factory.</p> Full article ">Figure 4
<p>Relative expression of TsV genes TsV_019 and TsV_014, encoding for putative DNA Polymerase (DNA Pol) and Capsid Decoration Protein (CDP), respectively. RGE, relative gene expression units.</p> Full article ">Figure 5
<p>Phylogenies obtained for TsV_01, TsV_18, and TsV_19 genes, respectively, accompanied by homologous sequences retrieved from PFAM. Branches indicate support values obtained with Maximum Likelihood aLRT SH-like algorithm implemented in Phylogeny.fr.</p> Full article ">
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Review
Bone Marrow Gene Therapy for HIV/AIDS
by Elena Herrera-Carrillo and Ben Berkhout
Viruses 2015, 7(7), 3910-3936; https://doi.org/10.3390/v7072804 - 17 Jul 2015
Cited by 19 | Viewed by 15868
Abstract
Bone marrow gene therapy remains an attractive option for treating chronic immunological diseases, including acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV). This technology combines the differentiation and expansion capacity of hematopoietic stem cells (HSCs) with long-term expression of therapeutic transgenes [...] Read more.
Bone marrow gene therapy remains an attractive option for treating chronic immunological diseases, including acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV). This technology combines the differentiation and expansion capacity of hematopoietic stem cells (HSCs) with long-term expression of therapeutic transgenes using integrating vectors. In this review we summarize the potential of bone marrow gene therapy for the treatment of HIV/AIDS. A broad range of antiviral strategies are discussed, with a particular focus on RNA-based therapies. The idea is to develop a durable gene therapy that lasts the life span of the infected individual, thus contrasting with daily drug regimens to suppress the virus. Different approaches have been proposed to target either the virus or cellular genes encoding co-factors that support virus replication. Some of these therapies have been tested in clinical trials, providing proof of principle that gene therapy is a safe option for treating HIV/AIDS. In this review several topics are discussed, ranging from the selection of the antiviral molecule and the viral target to the optimal vector system for gene delivery and the setup of appropriate preclinical test systems. The molecular mechanisms used to formulate a cure for HIV infection are described, including the latest antiviral strategies and their therapeutic applications. Finally, a potent combination of anti-HIV genes based on our own research program is described. Full article
(This article belongs to the Special Issue Gene Technology and Resistance to Viruses - Reviews)
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<p>Target cells for an anti-HIV gene therapy. Shown is the scheme of hematopoiesis. Either hematopoietic stem cells (HSCs) from bone marrow or the mature CD4<sup>+</sup> T cells can be targeted. These two cell populations are boxed.</p> Full article ">Figure 2
<p>Steps of the HIV-1 replication cycle that can be targeted by gene therapy. The HIV-1 replication steps that can be targeted by gene therapy are shown: (<b>1</b>) HIV-1 binding to cell membrane; (<b>2</b>) HIV-1 entry into the cell; (<b>3</b>) reverse transcription; (<b>4</b>) transport of the HIV-1 proviral genome into the nucleus; (<b>5</b>) integration of the viral genome into the cellular DNA; (<b>6</b>) transcription of the HIV-1 proviral genome; (<b>7</b>) translation of the viral messenger RNA (mRNA) into new viral proteins; (<b>8</b>) virion assembly inside the cell; and (<b>9</b>) maturation of the immature virion into a completely infectious particle.</p> Full article ">Figure 3
<p>The endogenous miRNA and exogenous shRNA processing pathways. The intracellular processing pathways are depicted starting from the miRNA gene of the cell (endogenous) or the transduced shRNA gene cassette (exogenous). The canonical Dicer-dependent and noncanonical Dicer-independent pathways are depicted for both molecules. Ago2 plays an essential role in Dicer-independent pathways. See the text for further details. PACT: Protein activator of protein kinase R; Pri-miRNA: Primary miRNA; Ago2: Argonaute 2 nuclease; RISC: RNA-induced silencing complex; TRBP: Transactivation response RNA-binding protein.</p> Full article ">Figure 4
<p>Combinatorial RNAi strategies. Four inhibitory scenarios are plotted with the respective advantages and disadvantages. This figure was adapted from [<a href="#B111-viruses-07-02804" class="html-bibr">111</a>]. LV: lentiviral vector.</p> Full article ">Figure 5
<p>Self-inactivating lentiviral vectors for stable shRNA expression. (<b>A</b>) The lentiviral vector JS1 is shown with three plasmids needed for lentiviral vector production. The vector genome is expressed from the Rous Sarcoma Virus (RSV) promoter. Transcripts start with the HIV-1 R and U5 regions and the packaging signal (ψ). The enhanced green fluorescent protein (GFP) reporter is expressed from the phosphoglycerate kinase promoter (PGK). Transcription of the vector genome and the GFP reporter terminates at the HIV-1 polyA signal within the 3′ LTR; (<b>B</b>) Scheme of a hematopoietic stem cell (HSC) clinical trial. An HIV-infected patient who fails on regular drug therapy will undergo apheresis for the collection of CD34<sup>+</sup> HSC after pretreatment with granulocyte-colony stimulatory factor (G-CSF). The mixed cell population containing CD34<sup>+</sup> HSC will be purified and transduced <span class="html-italic">ex vivo</span> with the therapeutic construct. Transduced cells will be infused back into the patient and the antiviral gene should protect these cells against HIV-1.</p> Full article ">
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Article
Optimization and Characterization of Candidate Strain for Coxsackievirus A16 Inactivated Vaccine
by Jingliang Li, Guanchen Liu, Xin Liu, Jiaxin Yang, Junliang Chang, Wenyan Zhang and Xiao-Fang Yu
Viruses 2015, 7(7), 3891-3909; https://doi.org/10.3390/v7072803 - 17 Jul 2015
Cited by 13 | Viewed by 7559
Abstract
Coxsackievirus A16 (CA16) and enterovirus 71 (EV71), both of which can cause hand, foot and mouth disease (HFMD), are responsible for large epidemics in Asian and Pacific areas. Although inactivated EV71 vaccines have completed testing in phase III clinical trials in Mainland China, [...] Read more.
Coxsackievirus A16 (CA16) and enterovirus 71 (EV71), both of which can cause hand, foot and mouth disease (HFMD), are responsible for large epidemics in Asian and Pacific areas. Although inactivated EV71 vaccines have completed testing in phase III clinical trials in Mainland China, CA16 vaccines are still under development. A Vero cell-based inactivated CA16 vaccine was developed by our group. Screening identified a CA16 vaccine strain (CC024) isolated from HFMD patients, which had broad cross-protective abilities and satisfied all requirements for vaccine production. Identification of the biological characteristics showed that the CA16CC024 strain had the highest titer (107.5 CCID50/mL) in Vero cells, which would benefit the development of an EV71/CA16 divalent vaccine. A potential vaccine manufacturing process was established, including the selection of optimal time for virus harvesting, membrane for diafiltration and concentration, gel-filtration chromatography for the down-stream virus purification and virus inactivation method. Altogether, the analyses suggested that the CC-16, a limiting dilution clone of the CC024 strain, with good genetic stability, high titer and broad-spectrum immunogenicity, would be the best candidate strain for a CA16 inactivated vaccine. Therefore, our study provides valuable information for the development of a Vero cell-based CA16 or EV71-CA16 divalent inactivated vaccine. Full article
(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Cross-neutralization assay of various CA16 isolated strains, SHZH05 and G10. (<b>a</b>) Immunization schedule for all virus strains; (<b>b</b>–<b>h</b>) Serum neutralization titers of CC024, CC045, CC090, CC097, CC163, SHZH05 and G10 against other CA16 and EV71, respectively. Neutralization titers (Y-axis) are plotted on a logarithmic scale.</p> Full article ">Figure 2
<p>Growth kinetics of various CA16 virus strains in Vero cells. Cells were infected at the MOI of 0.01, and the titer of the CC024 strain (10<sup>7.2</sup> CCID<sub>50</sub>/mL) was the highest among the seven viruses at 96 h post-infection</p> Full article ">Figure 3
<p>Biological characteristics of CC024 vaccine strain. (<b>a</b>) Concentrated virus was layered onto 20%–60% sucrose gradients and subjected to ultracentrifugation as described in Materials and Methods. Nineteen fractions were taken from the top to bottom and assayed. The odd fractions were analyzed by Western blot using a CA16 polyclonal antibody; (<b>b</b>) Viral RNAs of peaks at F9 and F14 were detected by RT-PCR; (<b>c</b>) Viral titers of peaks at F9 and F14 were detected in Vero cell lines; (<b>d</b>,<b>e</b>) Electron microscopy of CC024 virus from peaks at F9 and F14. Bar = 200 nm.</p> Full article ">Figure 4
<p>Growth curves of CC024, CC-05, CC-08, CC-11, CC-13 and CC-16 strains during passage in Vero cells infected at the MOI of 0.01. The titer of the CC-16 strain (10<sup>7.2</sup>CCID<sub>50</sub>/mL) was the highest at 96 h post-infection.</p> Full article ">Figure 5
<p>Neutralizing antibody titers of the 11th, 13th and 15th passages of CA16 CC-16. ICR mice (<span class="html-italic">n</span> = 6) were immunized with the same procedure and dose. Neutralization titers of the sera were determined as described in Materials and Methods and plotted (Y-axis) on a logarithmic scale.</p> Full article ">Figure 6
<p>Growth kinetics of CC-16 strain and images of infected Vero cells. (<b>a</b>) Confluent Vero cells in cell factories were infected with CA16 at the MOI of 0.0001, 0.001, 0.01 and 0.1. The highest titer was 10<sup>7.5</sup> CCID<sub>50</sub>/mL achieved with the MOI of 0.01; (<b>b</b>–<b>f</b>) Images of Vero cells infected with CC-16 on the fourth day post-infection; (<b>g</b>) Image of control Vero cells. Bar = 100 µm.</p> Full article ">Figure 7
<p>Gel filtration chromatography. (<b>a</b>) Elution profile of virus concentrate loaded and separated on the Sepharose Fast Flow 6 column using an AKTA system and monitored by UV absorption at 280 nm. The protein content in each collected fraction was separated and analyzed by Western blot using a CA16 polyclonal antibody (<b>b</b>) and silver staining (<b>c</b>).</p> Full article ">Figure 8
<p>The serum neutralizing antibody responses to CA16 viruses prepared with different inactivation methods. Groups of ICR mice (<span class="html-italic">n</span> = 6) were immunized with purified inactivated CA16 with or without adjuvant, or with adjuvant only as a control. Virus represents as inactivated by BPL and without adjuvant, BPL represents as inactivated by BPL and with adjuvant, Formalin represents as inactivated by formalin and with adjuvant Symbols represent reciprocal neutralizing antibody titers from groups of six animals. Neutralization titers (Y-axis) are plotted on a logarithmic scale.</p> Full article ">Figure 9
<p>Persistence of CA16 neutralizing antibody. (<b>a</b>) Immunization schedule and time points for testing of neutralizing antibody titers; (<b>b</b>) CA16 whole virus specific antibody responses were analyzed by measuring neutralizing antibody titers at the 4th, 6th, 8th, 10th and 12th week after primary immunization. Neutralization titers (Y-axis) are plotted on a logarithmic scale.</p> Full article ">
1637 KiB  
Review
Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia
by Nagayasu Egawa, Kiyofumi Egawa, Heather Griffin and John Doorbar
Viruses 2015, 7(7), 3863-3890; https://doi.org/10.3390/v7072802 - 16 Jul 2015
Cited by 395 | Viewed by 22004
Abstract
Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial [...] Read more.
Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted. Full article
(This article belongs to the Special Issue Tumour Viruses)
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<p>Evolutionary Relationship between Human Papillomaviruses. The human papillomaviruses types found in humans fall into five genera, with the Alpha-, Beta- (<b>blue</b>) and Gammapapillomavirus (<b>green</b>) representing the largest groups; Human papillomaviruses types from the Alphapapillomavirus genus are often classified as low-risk cutaneous (<b>light brown</b>); low-risk mucosal (<b>yellow</b>); or high-risk (<b>pink</b>) according to their association with the development of cancer. The high-risk types highlighted with red text are confirmed as “human carcinogens” on the basis of epidemiological data. The remaining high-risk types are “probable” or “possible” carcinogens. Although the predominant tissue associations of each genus are listed as either cutaneous or mucosal, these designations do not necessarily hold true for every member of the genus. The evolutionary tree is based on alignment of the <span class="html-italic">E1</span>, <span class="html-italic">E2</span>, <span class="html-italic">L1</span>, and <span class="html-italic">L2</span> genes [<a href="#B26-viruses-07-02802" class="html-bibr">26</a>]. HPV sequence data was be obtained from PaVE [<a href="#B1-viruses-07-02802" class="html-bibr">1</a>].</p> Full article ">Figure 2
<p>Viral gene-expression in adjacent Betapapillomavirus lesions. Immunostaining for the Betapapillomavirus E4 protein (<b>green</b>) reveals distinct patterns of expression in different lesions in an immunosuppressed individual. Lesion (<b>a</b>) and (<b>a`</b>) are atypical, and show marked basal and suprabasal staining; This pattern of E4 expression is distinct from typical E4 pattern of expression, which is usually restricted to the mid/upper epithelial layers as seen in region (<b>c</b>); Lesions (<b>b</b>) and (<b>b`</b>) show an intermediate pattern of E4 staining with expression close to the basal layer, as well as in the suprabasal cell layers (see also review [<a href="#B11-viruses-07-02802" class="html-bibr">11</a>]).</p> Full article ">Figure 3
<p>Alphapapillomavirus genome organization and the function of HPV proteins. (<b>A</b>) Genome organization typical of the high-risk Alphapapillomavirus types is illustrated by the genome of HPV16. The early (p97) and late (p670) promoters are marked by arrows. The six early ORFs (E1, E2, E4 and E5 (<b>in green</b>) and E6 and E7 (<b>in red</b>)) are expressed from the different promoters at different stages during epithelial cell differentiation. The late ORFs (L1 and L2 (<b>in yellow</b>)) are expressed from the p670 promoter in the upper epithelial layers as result of changes in splicing. The LCR/URR also contains the replication origin as well as post-transcriptional control sequences that contribute to viral gene expression. (<b>B</b>) The function of viral proteins. All known papillomavirus encodes a group of “core” proteins that were present early on during papillomavirus evolution, and which are conserved in sequence and in function between PV types. These include E1, E2, L2 and L1. The E4 protein may also be a core protein that has evolved to meet papillomaviruses epithelial specialization. The accessory proteins have evolved in each papillomavirus type during adaptation to different epithelial niches. The sequence and function of these genes are divergent between types. In general, these proteins are involved in modifying the cellular environment to facilitate virus life cycle completion, contributing to the virulence and pathogenicity. Knowledge of accessory protein function comes primarily from the study of Alphapapillomavirus types.</p> Full article ">Figure 4
<p>Regulation and deregulation of the high-risk Alphapapillomavirus life cycle. (<b>A</b>) The papillomavirus life cycle is regulated during epithelial cell differentiation and is shown diagrammatically. Cells that are driven through the cell cycle as a result of E6 and E7 expression are marked with red nuclei. The up-regulation of viral proteins necessary for genome amplification (<span class="html-italic">i.e.</span>, E1 and E2) requires activation of the viral late promoter in the upper epithelial layers (cells shown in green with red nuclei), with virus particles subsequently being released from the epithelial surface; (<b>B</b>) In HPV-associated neoplasia, late gene expression is retarded, and although the order of events remains the same, the production of infectious virions is restricted to smaller and smaller areas close to the epithelial surface. This situation is thought to be accompanied elevated E6/E7 expression, and represents a non-productive or poorly productive abortive infection. Integration of HPV DNA into the host cell genome is facilitated by deregulated E6/E7 expression. If integration disrupts the E1/E2 region this can allow the persistent high-level expression of E6 and E7 and the accumulation of genetic errors in the host genome. Eventually, the productive virus life cycle is no longer supported and viral episomes are lost (reviewed in [<a href="#B83-viruses-07-02802" class="html-bibr">83</a>]).</p> Full article ">Figure 5
<p>The difference of host cells (the site of infection) affects viral pathogenicity. Most cervical cancers arise at the cervical transformation zone. The transformation zone is maintained by a specialized type of tissue stem cell known as the reserve cell (shown in purple in the transformation zone), and possibly also by a cluster of cuboidal cells (<b>yellow</b>) localized more precisely at the squamo-columnar junction. These cells can maintain either the columnar epithelium of the endocervix or the stratified epithelium of transformation zone depending on their extracellular environment. In the ectocervix, the epithelium is populated by conventional epithelial tissue stem cells (purple in the ectocervix). The different characteristics of the various tissue stem cells that HPV infects are thought to influence the pattern of viral gene expression differently [<a href="#B41-viruses-07-02802" class="html-bibr">41</a>]. Current thinking suggests that productive infection is favoured at the ectocervix, while a non-productive or abortive infection is more likely at the endocervix. In the immunostains, MCM expression (<b>red</b>) indicates the expression of viral E7 protein. E4 expression (<b>green</b>) indicates the productive infection [<a href="#B59-viruses-07-02802" class="html-bibr">59</a>].</p> Full article ">Figure 6
<p>Epithelial tissue sites and tissue stem cells as targets for HPV infection. HPVs infect a variety of epithelial tissue sites, and can cause lesions in the vicinity of hair follicles, eccrine and apocrine sweat apparatus), nails, and also the inter-appendageal epidermis. Specialized epithelial sites contain other appendages, such as the salivary glands of the oral cavity and the tonsillar crypts of the oropharynx, where oropharyngeal cancers arise. The transformation zone regions, where stratified epithelium abuts columnar epithelium such as the cervical (<a href="#viruses-07-02802-f005" class="html-fig">Figure 5</a>) or anal transformation zones, are other target sites where infection is thought to be facilitated. (<b>A</b>) The bulge region of the hair follicle is a well characterized region where the stem cells that populate cutaneous epithelial sites reside. HPV virions are thought to gain access to the epithelial stem cells (coloured purple), either through a wound or possibly through the hair follicle; (<b>B</b>) Between the hair follicles, the tissue stem cells are thought to reside in both rete ridge and over the dermal papilla, and are not thought to be clustered at any specific location in the basal component [<a href="#B109-viruses-07-02802" class="html-bibr">109</a>]. With the sweat apparatus, at least two distinct stem cell populations have been identified that may be accessible for infection, either in the gland or the duct. These are able to repair damaged epidermis. HPV virions are thought to gain access to these stem cells (coloured purple), either through a wound or maybe through the eccrine duct; (<b>C</b>) The tonsillar crypts are a highly specialized lymphoepithelial tissue. A dense lymphocyte infiltrate generally obscures the junction between the lymphoid and epithelial components and splinters the epithelial sheath into irregular nests and cords. This reticulated epithelium may facilitate viral access to tissue stem cells at an immune-privileged site, which can inhibit virus-specific T cell activity and thereby facilitate immune evasion during initial HPV infection and subsequent virus-induced malignant transformation.</p> Full article ">Figure 7
<p>Examples for HPV targeting cutaneous appendages. (<b>A</b>) Haemotoxylin &amp; eosin stain (<b>left</b>) of a horizontal section of a tiny wart. An eccrine (Ec)-centered distribution of histological changes is observed. HPV1 DNAs are identified within the pathology changes following DNA <span class="html-italic">in situ</span> hybridization (<b>right</b>); (<b>B</b>) HPV63 DNA is identified in resident keratinocytes in the vicinity of eccrine ducts (Ec) in a ridge of the plantar skin following DNA <span class="html-italic">in situ</span> hybridization; (<b>C</b>) HPV6/11 histopathological changes are identified in the resident keratinocytes in and around the hair follicle (arrow) by haemotoxylin &amp; eosin staining (<b>left</b>) and DNA <span class="html-italic">in situ</span> hybridization (<b>right</b>).</p> Full article ">
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Commentary
A Viral Pilot for HCMV Navigation?
by Barbara Adler
Viruses 2015, 7(7), 3857-3862; https://doi.org/10.3390/v7072801 - 15 Jul 2015
Cited by 8 | Viewed by 4777
Abstract
gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric [...] Read more.
gH/gL virion envelope glycoprotein complexes of herpesviruses serve as entry complexes and mediate viral cell tropism. By binding additional viral proteins, gH/gL forms multimeric complexes which bind to specific host cell receptors. Both Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) express alternative multimeric gH/gL complexes. Relative amounts of these alternative complexes in the viral envelope determine which host cells are preferentially infected. Host cells of EBV can modulate the gH/gL complex complement of progeny viruses by cell type-dependent degradation of one of the associating proteins. Host cells of HCMV modulate the tropism of their virus progenies by releasing or not releasing virus populations with a specific gH/gL complex complement out of a heterogeneous pool of virions. The group of Jeremy Kamil has recently shown that the HCMV ER-resident protein UL148 controls integration of one of the HCMV gH/gL complexes into virions and thus creates a pool of virions which can be routed by different host cells. This first mechanistic insight into regulation of the gH/gL complex complement of HCMV progenies presents UL148 as a pilot candidate for HCMV navigation in its infected host. Full article
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<p>Model for a UL148-dependent regulation of alternative gH/gL complexes. HCMV strain-specific properties of the constituents of the gH/gL complexes determine the relative amounts of gH/gL/gO and gH/gL/UL128-131 complexes being formed and integrated into the viral envelope in the absence of UL148 (pink area). By reversibly binding to gH/gL, UL148 enhances binding of gH/gL to gO which results in an absolute increase in the amount of gH/gL/gO complexes in virions (blue area).</p> Full article ">Figure 2
<p>Model for a role of UL148 in shaping virus progenies. Based on differences in infection of fibroblasts and epithelial cells as described by Li <span class="html-italic">et al.</span> [<a href="#B10-viruses-07-02801" class="html-bibr">10</a>], a model of UL148-dependent shaping of virus progenies available for supernatant or cell-associated spread is proposed. Hypothetical and simplified virus progenies derived from fibroblasts infected with WT and ΔUL148 virus and the relative infectivities of these viral progenies are shown in (<b>A</b>); (<b>B</b>) shows a comparison of hypothetical virus progenies derived from fibroblasts and epithelial cells infected with WT or ΔUL148 virus. [gO/UL128] can infect fibroblasts and epithelial cells either cell-associated or via supernatant; [gO/−] can infect fibroblasts either cell-associated or via supernatant; [−/UL128] can infect fibroblasts and epithelial cells cell-associated, supernatant-driven infection of fibroblasts and epithelial cells is impaired; [−/−] not infectious.</p> Full article ">
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Review
Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems
by Marta Trevisan, Alessandro Sinigaglia, Giovanna Desole, Alessandro Berto, Monia Pacenti, Giorgio Palù and Luisa Barzon
Viruses 2015, 7(7), 3835-3856; https://doi.org/10.3390/v7072800 - 13 Jul 2015
Cited by 23 | Viewed by 7807
Abstract
The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of [...] Read more.
The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance. Full article
(This article belongs to the Special Issue Gene Technology and Resistance to Viruses - Reviews)
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<p>Representation of the workflow for the derivation of patient-specific induced pluripotent stem cells, their differentiation in somatic cells and tissues, and their use for disease modeling, drug screening, and development of personalized therapies.</p> Full article ">
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Article
Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance
by Chia-Wei Lin, Mei-Hsiu Su, Yu-Tsung Lin, Chien-Hung Chung and Hsin-Mei Ku
Viruses 2015, 7(7), 3816-3834; https://doi.org/10.3390/v7072799 - 9 Jul 2015
Cited by 4 | Viewed by 6493
Abstract
Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 [...] Read more.
Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. Full article
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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<p>Nucleotide sequence of <span class="html-italic">CmSPI</span>. The nucleotide sequence of <span class="html-italic">C. metuliferus</span> <span class="html-italic">CmSPI</span> gene, including the 5′-flanking region and 3′-UTR, is shown. The transcription start site is labeled +1, and the TATA box is shown in boldface. The core abscisic acid response element, T/GBOXATPIN2 element, and wound response element are boxed. Asterisk marks indicate the predicted reactive sites, K48 and D49. Flanking region sequences are shown in italic letters. Exon and intron predicted sequences are shown in upper and lower case letters, respectively.</p> Full article ">Figure 2
<p>Neighbor-joining reconstruction of the phylogenetic relationships among plant serine proteinases inhibitors. Bootstrap values for nodes supported in &gt;70% of 100 bootstrap replicates are shown above the branches. The scale bar shows the ratio of amino acid substitutions for a given horizontal branch length. The following reference sequences were included: <span class="html-italic">Solanum tuberosum</span> (CAA78259, AAZ08247, ACZ04396), <span class="html-italic">Solanum lycopersicum</span> (AAA34198, AAA60745), <span class="html-italic">Nicotiana tabacum</span> (CAA78269), <span class="html-italic">Nicotiana sylvestris</span> (AAA34067), <span class="html-italic">Arabidopsis lyrata</span> (EFH39906), <span class="html-italic">Vitis cinerea</span> (ADD51184), <span class="html-italic">Ricinus communis</span> (EEF41422), <span class="html-italic">Jatropha curcas</span> (ADB85100), <span class="html-italic">Salvia miltiorrhiza</span> (ABP01767), <span class="html-italic">Medicago truncatula</span> (AES61046), and <span class="html-italic">Populus trichocarpa</span> (EEF01895).</p> Full article ">Figure 3
<p>Constructs used for <span class="html-italic">C. metuliferus</span> and <span class="html-italic">N. benthamiana</span> transformation. (<b>A</b>) Construct for <span class="html-italic">C. metuliferus</span> <span class="html-italic">CmSPI</span> RNAi transformation. Partial sequence of <span class="html-italic">CmSPI</span> (203bp; from +46 to +692 nucleotide without intron sequences) was constructed into an invert-repeat form; (<b>B</b>) The construct for the transformation of full length genomic <span class="html-italic">CmSPI</span> into <span class="html-italic">N. benthamiana</span> plants. Arrows indicate the location of the primers used in the construction. RB, right border; Nos <span class="html-italic">pro</span>, nopaline synthase promoter; <span class="html-italic">NPT II</span>, neomycin phosphotransferase II; NOS <span class="html-italic">ter</span>, nopaline synthase terminator; CaMV 35S <span class="html-italic">enh</span>, cauliflower mosaic virus 35S enhancer; CaMV 35S <span class="html-italic">pro</span>, cauliflower mosaic virus 35S promoter; CaMV 35S <span class="html-italic">ter</span>, cauliflower mosaic virus 35S terminator; LB, left border.</p> Full article ">Figure 4
<p>Generation of <span class="html-italic">CmSPI</span> RNAi trangsenic <span class="html-italic">C. metuliferus</span>. (<b>A</b>) Southern hybridization analysis of DNA isolated from RNAi T<sub>0</sub> transgenic lines (T<sub>0</sub>H-1 to T<sub>0</sub>H-10) with α<sup>32</sup>P-labeled intron fragment probe. <span class="html-italic">C. metuliferus</span> line PI 292190 was used as a negative control; (<b>B</b>) PCR analysis of two RNAi transgenic T<sub>1</sub> lines (T<sub>1</sub>H-1 and T<sub>1</sub>H-3 lines) with intron specific primers. +, transgenic plants; –, non-transgenic plants; (<b>C</b>) Transcript levels of <span class="html-italic">CmSPI</span> in <span class="html-italic">C. metuliferus</span> lines (PRSV-resistant line PI 292190 and PRSV-susceptible line Acc. 2459) and two T<sub>1</sub> transgenic lines (T<sub>1</sub>H-1-2 and T<sub>1</sub>H-3-1). Total RNA from PRSV inoculated plants at 48 hpi was analyzed by RT-PCR. The ethidum bromide-stained rRNA is shown as a loading control; (<b>D</b>) Northern hybridization analysis of <span class="html-italic">CmSPI</span> in <span class="html-italic">C. metuliferus</span> lines (PI 292190 and Acc. 2459) and two T<sub>1</sub> transgenic plants (T<sub>1</sub>H-1-2 and T<sub>1</sub>H-3-1). Total RNA isolated from PRSV inoculated plants at 48 hpi and detected with α<sup>32</sup>P-labeled <span class="html-italic">CmSPI</span> probe. The ethidum bromide-stained rRNA is shown as a loading control; (<b>E</b>) Detection of short interfering RNAs in <span class="html-italic">C. metuliferus</span> lines (PI 292190 and Acc. 2459) and two T<sub>1</sub> transgenic plants (T<sub>1</sub>H-1-2 and T<sub>1</sub>H-3-1). Total RNA isolated from PRSV inoculated plants at 48 hpi and detected with α<sup>32</sup>P-labeled <span class="html-italic">CmSPI</span> probe. The ethidum bromide-stained rRNA is shown as a loading control; (<b>F</b>) CmSPI protein expression level in <span class="html-italic">C. metuliferus</span> lines (PI 292190 and Acc. 2459) and transgenic <span class="html-italic">CmSPI</span> RNAi plants (T<sub>1</sub>H-1-2 and T<sub>1</sub>H-3-1) by Western hybridization. Total soluble extracts from PRSV inoculated plants at 48 hpi and immunostaining with an anti-CmSPI polyclonal antibodies (CmSPI anti rabbit). The commassie blue-stained Rubisco is shown as a loading control.</p> Full article ">Figure 5
<p>Effect of <span class="html-italic">CmSPI</span> gene silencing on PRSV inoculated <span class="html-italic">C. metuliferus</span> plants. (<b>A</b>) PRSV symptoms on the whole plant are shown for <span class="html-italic">C. metuliferus</span> reistance line PI 292190, susceptible line Acc. 2459, <span class="html-italic">CmSPI</span> RNAi transgenic line T<sub>1</sub>H-1-2, and <span class="html-italic">CmSPI</span> RNAi transgenic line T<sub>1</sub>H-3-1 (from left to right); PRSV symptoms on leaves for (<b>B</b>) PI 292190; (<b>C</b>) Acc. 2459; (<b>D</b>) transgenic line T<sub>1</sub>H-1-2; and (<b>E</b>) transgenic line T<sub>1</sub>H-3-1.</p> Full article ">Figure 6
<p>PVY resistance of <span class="html-italic">CmSPI</span> T<sub>1</sub> transgenic <span class="html-italic">N. benthamiana</span> plants at 14 dpi. (<b>A</b>) Wild type <span class="html-italic">N. benthamiana</span>-mock; (<b>B</b>) Wild type <span class="html-italic">N. benthamiana</span>-PVY inoculated; (<b>C</b>) T<sub>1</sub>F-21-2- PVY inoculated; (<b>D</b>) T<sub>1</sub>F-51-10- PVY inoculated. Arrow indicates the symptoms of PVY on <span class="html-italic">N. benthamiana</span>.</p> Full article ">Figure 7
<p>Southern hybridization and RT-PCR analysis of <span class="html-italic">CmSPI</span> T<sub>2</sub> transgenic <span class="html-italic">N. benthamiana</span> lines. (<b>A</b>) Southern hybridization analysis of DNA isolated from wild type <span class="html-italic">N. benthamiana</span> control (WT), <span class="html-italic">CmSPI</span> T<sub>2</sub> transgenic lines, T<sub>2</sub>F-21-2-5 and T<sub>2</sub>F-51-10-8, with α<sup>32</sup>P-labeled <span class="html-italic">CmSPI</span> probe. <span class="html-italic">CmSPI</span> transformation plasmid DNA was used as a postive control; (<b>B</b>) Transcript levels of <span class="html-italic">CmSPI</span> in postive control (<span class="html-italic">C. metuliferus</span> resistance line PI 292190), negative control WT (wild type <span class="html-italic">N. benthamiana</span> plant), and <span class="html-italic">CmSPI</span> T<sub>2</sub> transgenic lines (T<sub>2</sub>F-21-2-5 and T<sub>2</sub>F-51-10-8). Total RNA from PVY inoculated plants at 2 dpi was analyzed by RT-PCR. The ethidum bromide-stained rRNA is shown as a loading control.</p> Full article ">
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Article
The Apis mellifera Filamentous Virus Genome
by Laurent Gauthier, Scott Cornman, Ulrike Hartmann, François Cousserans, Jay D. Evans, Joachim R. De Miranda and Peter Neumann
Viruses 2015, 7(7), 3798-3815; https://doi.org/10.3390/v7072798 - 9 Jul 2015
Cited by 67 | Viewed by 11326
Abstract
A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames [...] Read more.
A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family. Full article
(This article belongs to the Special Issue Honeybee Viruses)
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<p>Morphology of AmFV virions: (<b>A</b>,<b>B</b>) Electron micrographs of “milky” bee hemolymph containing characteristic AmFV nucleoproteins (np) and enveloped virions (v). Approximate size is indicated by scale bars.</p> Full article ">Figure 2
<p>Schematic representation of the AmFV genome. Characteristic ORFs presenting similarities to other large dsDNA viruses are indicated. Colors refer to putative functions: DNA replication and nucleotides metabolism genes (red), <span class="html-italic">pif</span> and <span class="html-italic">p74</span> homologs (green), <span class="html-italic">Bro</span> genes (blue), and putative virulence genes (black). The brown circle fragments correspond to the positions of the larger contigs (&gt;8500 bp) filtered out from <span class="html-italic">V. destructor</span> metagenome assembly and assigned initially as baculovirus-like sequences [<a href="#B20-viruses-07-02798" class="html-bibr">20</a>]. The inner circle represents the GC plot (above average: gold; below average: purple). The grey triangles flanked by arrows indicate unresolved sequences.</p> Full article ">Figure 3
<p>Multiple sequence alignments and phylogenetic trees for the DNA polymerase (<b>A</b>) and Bro-N (<b>B</b>) domains. Branch support values are indicated in red. GenBank entries from top to bottom: A, gi|365199561|, gi|1063688|, gi|409978642|, gi|589287870|, gi|9964364|, gi|15021419|, gi|187903038|, gi|312233904|, gi|2702251|, and gi|311977705|; and B, gi|325152622|, gi|21668326|, gi|55416627|, gi|9631452|, gi|672972322|, gi|736994060|, and gi|660515707|.</p> Full article ">Figure 4
<p>Phylogenetic analyses. Threes were built from thymidylate synthase (TS), ribonucleotide reductase (RR) and p74 proteins. Branch support values are indicated in red. GenBank entries from top to bottom: TS, gi|589287845|, gi|485725392|, gi|187903049|, gi|370702981|, gi|9631429|, gi|551484996|, gi|417072295|, and gi|55417112|; RR large domain, gi|15021484|, gi|347481982|, gi|370702993|, gi|294471329|, gi|22164662|, gi|422933669|, gi|187903102|; p74, gi|187903076|, gi|17016513|, gi|9630072|, gi|370703052|, and gi|531034105|.</p> Full article ">Figure 5
<p>Agarose gel electrophoresis showing AmFV PCR amplicons produced from different individuals (larvae, pupae or adults bees), queen bee tissues, drone sperm or bee products, using three different primer pairs (the thymidylate synthase (TS) primers were described by Cornman <span class="html-italic">et al.</span> (2010) [<a href="#B20-viruses-07-02798" class="html-bibr">20</a>]).</p> Full article ">
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Essay
Learning from Ebola Virus: How to Prevent Future Epidemics
by Alexander S. Kekulé
Viruses 2015, 7(7), 3789-3797; https://doi.org/10.3390/v7072797 - 9 Jul 2015
Cited by 14 | Viewed by 12595
Abstract
The recent Ebola virus disease (EVD) epidemic in Guinea, Liberia and Sierra Leone demonstrated that the World Health Organization (WHO) is incapable to control outbreaks of infectious diseases in less developed regions of the world. This essay analyses the causes for the failure [...] Read more.
The recent Ebola virus disease (EVD) epidemic in Guinea, Liberia and Sierra Leone demonstrated that the World Health Organization (WHO) is incapable to control outbreaks of infectious diseases in less developed regions of the world. This essay analyses the causes for the failure of the international response and proposes four measures to improve resilience, early detection and response to future outbreaks of infectious diseases. Full article
(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)
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Review
Cloned Defective Interfering Influenza RNA and a Possible Pan-Specific Treatment of Respiratory Virus Diseases
by Nigel J. Dimmock and Andrew J. Easton
Viruses 2015, 7(7), 3768-3788; https://doi.org/10.3390/v7072796 - 8 Jul 2015
Cited by 49 | Viewed by 7590
Abstract
Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of [...] Read more.
Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases in vivo using as an example a highly active cloned influenza A DI genome and virus that protects broadly in preclinical trials against different subtypes of influenza A and against non-influenza A respiratory viruses. This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials. Full article
(This article belongs to the Special Issue Gene Technology and Resistance to Viruses - Reviews)
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<p>Duration of residence of 244 RNA in the lungs of mice inoculated with 12 µg 244 DI virus protein, and prediction of the duration of protection from infectious challenge virus. The amount of DI RNA was determined by quantitative PCR (▲) (adapted from [<a href="#B2-viruses-07-02796" class="html-bibr">2</a>]). ▼, PCR signal from lungs of mice that were inoculated with diluent. Mice treated with 1.2 µg 244 DI virus were protected from lethal challenge with infectious IAV after 7 days had elapsed. However, 244 DI virus did not protect at 14 days after treatment, indicating that the DI RNA had declined to a sub-protective level and that no anti-DI virus immunity had been generated. The solid red line shows the amount and decline of the DI RNA in the lower DI virus dose, assuming that the amount of DI RNA in the lung and the kinetics of decline were proportional to the higher dose (in black). The red dashed line is an extrapolation from the 1.2 µg dose that was still protective after 7 days and suggests that the 12 µg dose of 244 DI virus would still be protective at 5 to 6 weeks after treatment. It is calculated that DI RNA from the high dose declines to zero in approximately 6 months.</p> Full article ">
830 KiB  
Review
Tissue Barriers to Arbovirus Infection in Mosquitoes
by Alexander W. E. Franz, Asher M. Kantor, A. Lorena Passarelli and Rollie J. Clem
Viruses 2015, 7(7), 3741-3767; https://doi.org/10.3390/v7072795 - 8 Jul 2015
Cited by 293 | Viewed by 19633
Abstract
Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, [...] Read more.
Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts. Full article
(This article belongs to the Special Issue Interactions between Arboviruses and Arthropod Vectors)
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Graphical abstract
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<p>Persistent arbovirus infection of a mosquito vector requires successful crossing of tissue barriers by the virions. (<b>A</b>) Schematic representation of arbovirus tropism in a mosquito vector [after Snodgrass [<a href="#B21-viruses-07-02795" class="html-bibr">21</a>], modified]. Virions are represented by blue hexagons. (<b>B</b>) Schematic representation of a permissive midgut infection, midgut infection barrier (MIB) and midgut escape barrier (MEB). Grey squares represent midgut epithelial cells and blue hexagons represent virions. Images below show the presence of antigen of DENV2-Jamaica 1409 (green) in midgut (at 7 days post-bloodmeal) or salivary glands (at 14 days post-bloodmeal) of <span class="html-italic">Aedes aegypti</span>, as detected by immunofluorescence assay using DENV2-specific monoclonal antibody 3H5. Tissues were counter-stained with Evans blue. Images were viewed under a fluorescent microscope equipped with FITC-specific filter sets. MG, midgut; SG, salivary gland.</p> Full article ">Figure 2
<p>Ultrastructural views of the posterior midgut tissue of <span class="html-italic">Ae. aegypti</span>. (<b>A</b>) Cross-section of a non-infected midgut of a female at 7 days post-bloodmeal (magnification: 600×). (<b>B</b>) Close-up view of image in panel (<b>A</b>) at 2000× magnification. Note the structured BL surrounding the midgut. Dissected midguts were fixed in 2% glutaraldehyde, 2% paraformaldehyde fixative followed by embedding in histogel and post-fixation in 1% osmium tetroxide. A dehydration series in ethanol was performed prior to embedding of midguts in Epon/Spurs resin. Resin-embedded midguts were thin-sectioned and stained with lead citrate. Images were captured using a JEOL 1400 transmission electron microscope.</p> Full article ">Figure 3
<p>Ultrastructural view of the posterior midgut tissue of an <span class="html-italic">Ae. aegypti</span> female orally infected with CHIKV 37997 at 7 days post-infection. Virus titer in the bloodmeal was ~10<sup>7</sup> pfu/mL. CHIKV virions are present only in the basal labyrinth of the epithelial cell, which is the putative site of viral assembly. Virions are visible budding from the basal plasma membrane in close proximity to the BL. Virions crossing the BL were not observed (magnification: 8000×).</p> Full article ">
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Review
Adenovirus 36 and Obesity: An Overview
by Eleonora Ponterio and Lucio Gnessi
Viruses 2015, 7(7), 3719-3740; https://doi.org/10.3390/v7072787 - 8 Jul 2015
Cited by 47 | Viewed by 14983
Abstract
There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases [...] Read more.
There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed. Full article
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<p>Genomic organization of Adv36: The early proteins (E1A, E1B, E2A, E2B, E3, and E4) are involved in the regulation of replication of DNA. The late proteins (L1–L5), products of the translation of late mRNA, constitute structural capsid proteins.</p> Full article ">Figure 2
<p>Adv36 mediates glucose uptake independently from insulin, adapted from [<a href="#B64-viruses-07-02787" class="html-bibr">64</a>]. Adv36 up-regulates the Phosphoinositide 3-kinase (PI3K) signaling via Ras, increasing cellular glucose uptake by glucose trasporters Glut1 and Glut4 despite a down-regulation of the Insulin Receptor Substrate (IRS) signaling.</p> Full article ">Figure 3
<p>Adv36 and leptin. (<b>A</b>) Leptin binding to its receptor (ObRb) activates the associated JAK-2 tyrosine kinase. Leptin inhibits glucose transport through GLUT-2, and activates PI3K. Additionally, Phosphoinositide 3-kinase (PI3K) activation by leptin reduces Cyclic adenosine monophosphate (cAMP) levels and activates the Protein kinase A (PKA) pathway. Leptin can also inhibit the phospholipase C (PLC)/protein kinase C (PKC) pathway; (<b>B</b>) Adv36 inhibits leptin production. The results are decreased insulin release and increased lipid accumulation.</p> Full article ">Figure 4
<p>Proposed mechanisms underlying the effects of Adv36 in infected individuals.</p> Full article ">
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Review
Resistance against Integrase Strand Transfer Inhibitors and Relevance to HIV Persistence
by Thibault Mesplède and Mark A. Wainberg
Viruses 2015, 7(7), 3703-3718; https://doi.org/10.3390/v7072790 - 7 Jul 2015
Cited by 45 | Viewed by 7682
Abstract
Drug resistance prevents the successful treatment of HIV-positive individuals by decreasing viral sensitivity to a drug or a class of drugs. In addition to transmitted resistant viruses, treatment-naïve individuals can be confronted with the problem of drug resistance through de novo emergence of [...] Read more.
Drug resistance prevents the successful treatment of HIV-positive individuals by decreasing viral sensitivity to a drug or a class of drugs. In addition to transmitted resistant viruses, treatment-naïve individuals can be confronted with the problem of drug resistance through de novo emergence of such variants. Resistant viruses have been reported for every antiretroviral drug tested so far, including the integrase strand transfer inhibitors raltegravir, elvitegravir and dolutegravir. However, de novo resistant variants against dolutegravir have been found in treatment-experienced but not in treatment-naïve individuals, a characteristic that is unique amongst antiretroviral drugs. We review here the issue of drug resistance against integrase strand transfer inhibitors as well as both pre-clinical and clinical studies that have led to the identification of the R263K mutation in integrase as a signature resistance substitution for dolutegravir. We also discuss how the topic of drug resistance against integrase strand transfer inhibitors may have relevance in regard to the nature of the HIV reservoir and possible HIV curative strategies. Full article
(This article belongs to the Special Issue Gene Technology and Resistance to Viruses - Reviews)
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<p>HIV resistant viruses are archived within the viral reservoir upon treatment failure and can rapidly reemerge following treatment re-initiation.</p> Full article ">Figure 2
<p>Dolutegravir may inhibit HIV resistant viruses from becoming archived within the viral reservoir.</p> Full article ">
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Review
Resistance to Rhabdoviridae Infection and Subversion of Antiviral Responses
by Danielle Blondel, Ghizlane Maarifi, Sébastien Nisole and Mounira K. Chelbi-Alix
Viruses 2015, 7(7), 3675-3702; https://doi.org/10.3390/v7072794 - 7 Jul 2015
Cited by 24 | Viewed by 11598
Abstract
Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play [...] Read more.
Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play a role in mediating the antiviral response in vivo using gene knockout mouse models. IFNs inhibit rhabdovirus replication at different stages via the induction of a variety of ISGs. This review will discuss how individual ISG products confer resistance to rhabdoviruses by blocking viral entry, degrading single stranded viral RNA, inhibiting viral translation or preventing release of virions from the cell. Furthermore, this review will highlight how these viruses counteract the host IFN system. Full article
(This article belongs to the Special Issue Gene Technology and Resistance to Viruses - Reviews)
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<p>The different steps of the rhabdovirus cycle inhibited by interferon (IFN)-stimulated gene (ISG) products. IFN-inducible transmembrane (IFITM) proteins block viral entry, CH25h impairs the virus cell-fusion step by inducing cellular membrane changes, MxA inhibits primary transcription, ISG20 and ProMyelocytic Leukemia (PML) inhibit secondary transcription, protein kinase (PKR) and IFIT proteins inhibit viral translation and Tetherin prevents release of virions from the cell.</p> Full article ">Figure 2
<p>Innate immune sensing of rhabdovirus infection. In rhabdovirus-infected cells, the viral RNA is mainly detected by RIG-I. Once activated, RIG-I binds to the CARD containing adaptor protein IPS-1 (also known as MAVS, CARDIF or VISA), which phosphorylates IRF3 and/or IRF7 through TRAF3, NAP1 and TBK1/IKKε. Phosphorylated IRF3 and IRF7 homodimerize and translocate into the nucleus where they induce the expression of Type I IFN genes. IPS-1 also interacts with FADD, a death domain-containing adapter involved in death receptor signaling, and RIP1, which induces the activation of the NF-κB pathway. NF-κB is composed of homo- and heterodimeric complexes of members of the Rel family. The most common and best-characterized form of NF-κB is the p65/p50 heterodimer. A new member RelAp43 (p43) of the NF-κB family has been recently identified. Activation of the IκB kinase (IKK) complex, consisting of catalytic kinase subunits (IKKα and/or IKKβ) and the regulatory non-enzymatic scaffold protein NEMO, results in the phosphorylation and subsequence degradation of IκB. This enables free NF-κB to translocate to the nucleus, where it induces target gene expression, including pro-inflammatory cytokine encoding genes. The steps inhibited by the RABV-N and -P proteins, as well as VSV M protein are indicated in the diagram above.</p> Full article ">Figure 3
<p>Inhibition of IFN signaling by RABV-P. The interaction of Type I IFN with IFNAR leads to the activation of the JAK tyrosine kinases (Tyk2 and JAK1) resulting in the phosphorylation of STAT1 and STAT2, which form, with IRF9, the complex ISGF3. ISGF3 translocates to the nucleus and induces the expression of ISGs that harbor an ISRE. The binding of Type II IFN to its receptor, IFNGR, results in the phosphorylation of STAT1 by JAK1 and JAK2. pSTAT1 homodimers migrate to the nucleus and bind to the GAS in the promoter region of specific ISGs. The steps counteracted by the RABV-P are indicated: P interacts with STAT1 and STAT2, and thereby blocks IFN signaling by STAT1 sequestration in the cytoplasm and the inhibition of pSTAT1 and ISGF3 binding to DNA promoters.</p> Full article ">Figure 4
<p>Functional and structural characterization of RABV-P protein. The RABV-P gene encodes a full length (P1) and four N-terminally truncated isoforms (P2-P5) from the initial and subsequent internal, in frame Met codons at indicated residues. RABV-P protein contains three functional domains separated by two intrinsically disordered regions: the N-terminal domain (<span class="html-italic">P-NTD</span>, residues 1 to 52), interacting with the soluble N protein (called N°) and L protein; the dimerization domain (<span class="html-italic">Pdim</span>, residues 91 to 131); and the C-terminal domain (<span class="html-italic">P-CTD</span>, residues 186 to 297), involved in binding to the N-RNA, as well as cellular proteins STAT1 and PML. The P protein also contains several targeting sequences including two NESs, a conformational NLSc, formed by the globular fold of the CTD.</p> Full article ">
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Review
Early Events in Chikungunya Virus Infection—From Virus CellBinding to Membrane Fusion
by Mareike K. S. Van Duijl-Richter, Tabitha E. Hoornweg, Izabela A. Rodenhuis-Zybert and Jolanda M. Smit
Viruses 2015, 7(7), 3647-3674; https://doi.org/10.3390/v7072792 - 7 Jul 2015
Cited by 96 | Viewed by 13118
Abstract
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV [...] Read more.
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research. Full article
(This article belongs to the Special Issue Viruses and Exosomes)
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<p>Structure of the E2/E1 dimer. (<b>A</b>) Ribbon diagram showing the ectodomains of the CHIKV E1 and E2 glycoprotein ([<a href="#B22-viruses-07-02792" class="html-bibr">22</a>]; PDB 3N41). The structural domains I, II, and III of E1 are shown in blue, red and yellow, respectively. E2 domain A, B, and C are designated in cyan, green, and pink, respectively. In the mature virion, the E1 fusion loop (E1-FL, orange), is covered by a binding groove between E2 domain A and B. The β-ribbon of E2 containing the acid-sensitive region is highlighted in dark purple. Within this region, the hydrogen bond between E2-H170 and E1-S57 stabilizes the E2/E1 dimer interaction at neutral pH [<a href="#B26-viruses-07-02792" class="html-bibr">26</a>,<a href="#B142-viruses-07-02792" class="html-bibr">142</a>]. E1-A/V226 and E1-V178 are important for lipid sensing before fusion [<a href="#B12-viruses-07-02792" class="html-bibr">12</a>,<a href="#B154-viruses-07-02792" class="html-bibr">154</a>]. The black arrow points towards the viral membrane; (<b>B</b>,<b>C</b>) Surface view (PDB 2XFC) of one virus spike from the side (<b>B</b>) and the top (<b>C</b>). E1 is depicted in the same colors as in the ribbon diagram, E2 is depicted in gray for clarity. This figure was prepared using the program PyMOL.</p> Full article ">Figure 2
<p>Chikungunya virus cell entry and potential antiviral strategies. The viral life cycle starts with attachment of the virus particle to one of the ubiquitously expressed attachment factors or receptors at the cell surface (<b>1</b>); Subsequently, the virus is internalized into the cell via clathrin-mediated endocytosis (<b>2</b>); Then, clathrin-molecules dissociate from the vesicle and the virus is delivered to Rab5<sup>+</sup> endosomes. Within the mildly acidic lumen of the endosome, the viral glycoproteins E2 and E1 undergo major conformational changes that lead to membrane fusion (<b>3</b>); Thereafter, the nucleocapsid core is released into the cytosol (<b>4</b>). The molecules and compounds that are known to interfere with entry are stated in the boxes.</p> Full article ">Figure 3
<p>Model of alphavirus membrane fusion [<a href="#B145-viruses-07-02792" class="html-bibr">145</a>]. (<b>a</b>) On a mature virion, 240 copies of E1 and E2 are arranged as 80 trimeric spikes; a single spike consisting of three E2/E1 heterodimers. Domains of E1 are colored as in figure 1; E2 is shown in gray. The E1 hydrophobic fusion loop (indicated as a star) is buried in a groove between domain A and domain B of E2; (<b>b</b>) Destabilization of the E2/E1 heterodimer is triggered once the virus is exposed to the mildly acidic pH. Domain B of E2 moves away and the E1 fusion loop is exposed; (<b>c</b>) Insertion of the fusion loop into the target membrane and dissociation of the E2 protein. Formation of a E1 core trimer between DI and DII; (<b>d</b>,<b>e</b>) Re-folding of E1 DIII and stem region to form a hairpin-like homotrimer, forcing the two opposing membranes together; (<b>f</b>) Merging of the opposing membrane leaflets (hemifusion); (<b>g</b>) Formation of the final stable homotrimer and opening of the fusion pore. (Figure reprinted with permission from Nature Reviews Microbiology).</p> Full article ">
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Article
Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations
by Aurélien Chateigner, Annie Bézier, Carole Labrousse, Davy Jiolle, Valérie Barbe and Elisabeth A. Herniou
Viruses 2015, 7(7), 3625-3646; https://doi.org/10.3390/v7072788 - 7 Jul 2015
Cited by 51 | Viewed by 9953
Abstract
Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a [...] Read more.
Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%). K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs). Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential. Full article
(This article belongs to the Special Issue Next Generation Sequencing: New Developments and Discoveries in Virology)
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Graphical abstract

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<p><span class="html-italic">Autographa californica multiple nucleopolyhedrovirus</span> (AcMNPV) occlusion bodies. (<b>A</b>) Scanning electron microscopy (×20,000) showing several occlusion body (OB) shapes. The silhouette of virions is visible on emptied OBs (E); (<b>B</b>) Transmission electron microscopy (×50,000) showing the cross section of one OB with rod shape virions (V) and nucleocapsids (NC).</p> Full article ">Figure 2
<p>Strategy for finding large deletions. A genome consensus sequence section is shown along with the two pair reads associated with it and the position they map depending on the analyses conducted. Grey bars between reads and the consensus sequence represent a good alignment. (<b>1</b>) Read 1 and Read 2 represent paired-end reads, they should theoretically map on the consensus sequence with an overlap of about 42 bp as the insert is 260 bp long; (<b>2</b>) The actual mapping with a larger distance between reads of a pair and/or a poor mapping of the end of the reads can differ from expectation based on the consensus; (<b>3</b>) This different mapping happens because the genome from which the reads were produced carried a deletion.</p> Full article ">Figure 3
<p>Location and frequency of non-consensus single nucleotide polymorphisms (SNPs) on the AcMNPV-WP10 consensus genome. The WP10 consensus genome is presented as a linear map. Arrows indicate the transcriptional direction of predicted ORFs. Arrows are colored according to the comparison of the WP10 and C6 AcMNPV genomes (green: ORFs with identical size in both genomes; pink: ORF fusion; grey: longer ORF, yellow: shorter ORF; see <a href="#viruses-07-02788-t001" class="html-table">Table 1</a> for details). Non-consensus SNPs are plotted as frequency at the locus they were identified with a color corresponding to the k-means cluster they belong to (<a href="#viruses-07-02788-t002" class="html-table">Table 2</a>). Stars highlight cluster 4 SNPs changing amino acid polarity.</p> Full article ">Figure 4
<p>Frequency of K-means clusters. For each violin plot, the white dot represents the median, the black bar limits represent the 1st and 3rd quartile (respectively the lower and the upper limit of the bar). The shapes of the violins represent the probability density of the nucleotides in the cluster for the different percentages. The color of the shading represents the nucleotide as shown in the legend (always in the order A: Adenine, T: Thymine, G: Guanine, C: Cytosine).</p> Full article ">Figure 5
<p>Deletion reads coverage along the AcMNPV-WP10 genome. The 5% (in black) and 2.81% (in red) reads presenting the highest pair distance were mapped on the genome. The coverage by these reads is shown all along the WP10 consensus genome, revealing deletion hotspots. The triangles represent the positions of the <span class="html-italic">hrs</span> on the genome. <span class="html-italic">Hrs</span> with their name under the triangle have been studied in more depth. The 2.81% reads are more distant than 669 nt that is the length of <span class="html-italic">hr2</span>, the largest <span class="html-italic">hr</span>. By comparing these two sets, we show that not only deletions of palindrome repeats in the <span class="html-italic">hrs</span> are present in the population, but also larger deletions, possibly occurring between two <span class="html-italic">hrs</span>.</p> Full article ">
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Review
Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence
by Alexandre Carpentier, Pierre-Yves Barez, Malik Hamaidia, Hélène Gazon, Alix De Brogniez, Srikanth Perike, Nicolas Gillet and Luc Willems
Viruses 2015, 7(7), 3603-3624; https://doi.org/10.3390/v7072793 - 7 Jul 2015
Cited by 43 | Viewed by 10904
Abstract
Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a [...] Read more.
Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. Full article
(This article belongs to the Special Issue Viral Replication Complexes: Structures, Functions, Applications and Inhibitors)
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<p>Model of HTLV-1 replication (<b>a</b>) HTLV-1 transmission occurs by breastfeeding, sexual intercourse, or blood transfusion. Except for blood transfer, initial infection requires crossing of the mucosal barrier by several mechanisms: (i) transmigration of HTLV-1 infected macrophages, (ii) transcytosis of viral particles, (iii) release of newly produced virions from the basal surface of infected epithelial cell, (iv) bypass of HTLV-1 infected cells through a damaged mucosa. HTLV-1 can then infect mucosal immune cells directly (cis-infection) or via antigen-presenting cells (APCs); (<b>b</b>) APCs can either become infected or transfer membrane-bound extracellular virions to T-cells (trans-infection). Cell-to-cell transfer of virions involves different non-exclusive mechanisms: a virological synapse, cellular conduits, or extracellular viral assemblies. Infection of resident cells occurs either in the mucosa or in secondary lymphoid organs. Soon after primary infection, HTLV-1 replicates by cell-to-cell infection (<span class="html-italic">i.e.</span>, the infectious cycle) or (<b>c</b>) by mitotic division of a cell containing an integrated provirus (clonal expansion). Since an antiviral immune response is quickly initiated, the efficacy of the infectious cycle is severely dampened down soon after infection.</p> Full article ">Figure 2
<p>Tax and HBZ promote proliferation and persistence of the infected cell. Tax activates survival pathways (CREB/Akt/NFkB), promotes mitosis (CDKs), and inhibits tumor suppressors (p53, TP53INP1, Bcl11B). Tax-mediated growth-promoting activities are counteracted by HBZ, mitigating unrestrained proliferation. The host immune response further controls infected cell proliferation. Tax-induced proliferation creates replicative stress and generates reactive oxygen species (ROS). Tax interacts with the mitotic checkpoint control protein Mad1 thereby inducing clastogenic damage. Tax attenuates the DNA damage response (DDR) induced by unscheduled cell proliferation. Inhibition of the DDR allows cells to accumulate DNA lesions and stabilize mutations. If uncontrolled by senescence or cell death mechanisms, growth-promoting mutations pave the way to disease development.</p> Full article ">
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Article
Genome Characterization, Prevalence and Distribution of a Macula-Like Virus from Apis mellifera and Varroa destructor
by Joachim R. De Miranda, R. Scott Cornman, Jay D. Evans, Emilia Semberg, Nizar Haddad, Peter Neumann and Laurent Gauthier
Viruses 2015, 7(7), 3586-3602; https://doi.org/10.3390/v7072789 - 6 Jul 2015
Cited by 53 | Viewed by 9146
Abstract
Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from [...] Read more.
Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV). Full article
(This article belongs to the Special Issue Honeybee Viruses)
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Graphical abstract
Full article ">Figure 1
<p>Genome organization of BeeMLV. The estimated genome size is indicated as are the presence of a natural 3′ poly-A tail and the location of overlapping open reading frames coding for the capsid protein (CP), an unknown 15 kD protein (P15) and the large polyprotein containing the domains MTR (methyl transferase), PRR (Proline-rich region), P-Pro (endo-peptidase), Helic (NTPase/helicase) and RdRp (RNA-dependent RNA polymerase), separated by the location of the putative P-Pro cleavage site in between the P-Pro and Helic domains. Additionally indicated are the location and sequence of the putative BeeMLV sub-genomic RNA promoter, compared to its homologues for the Tymo- and Marifiviruses. The lines above the genome map identify the locations of the nucleotide (<span class="html-italic">a</span>) and RdRp (<span class="html-italic">b</span>) plus CP (<span class="html-italic">c</span>) amino acid sequences used in the phylogenetic analyses (light grey) and of the quantitative RT-qPCR assays for either genomic (<span class="html-italic">d</span>, <span class="html-italic">e</span>) or sub-genomic + genomic (<span class="html-italic">f</span>, <span class="html-italic">g</span>) virus RNA quantification (dark grey).</p> Full article ">Figure 2
<p>(<b>A</b>) Unrooted Minimum Evolution phylograms describing the relationship between BeeMLV and other viruses in the <span class="html-italic">Tymoviridae,</span> based on the RNA-dependent RNA polymerase and capsid protein amino acid sequences. The scale bar represents the inferred evolutionary distance in amino acid substitutions per site. Bootstrap support for the various nodes is indicated by the white (&gt;55%), grey (&gt;70%) and black (&gt;85%) circles. The Tymovirus, Marafivirus and Maculavirus genera are marked with grey-shaded areas. The two insect-infecting <span class="html-italic">Tymoviridae</span>; <span class="html-italic">Bombyx mori</span> Macula-like latent virus (BmMLV) and Culex Tymoviridae-like virus (CuTLV) are indicated in both phylograms, as well as a second Tymoviridae-like virus (VTLV) recovered in these studies; (<b>B</b>) Unrooted Minimum Evolution phylogram describing the relationship between different geographic and biological BeeMLV isolates, based on the capsid protein nucleotide sequence. The scale bar represents the inferred evolutionary distance in nucleotide substitutions per site. Bootstrap support is indicated as for <a href="#viruses-07-02789-f002" class="html-fig">Figure 2</a>A. The taxa are consensus nucleotide sequences from Europe, Jordan, the USA and the pupal (Pu), adult (Ad) and varroa (V) pooled samples from apiaries in northern France (#28) and southern France (#878).</p> Full article ">Figure 3
<p>(<b>A</b>) Prevalence of BeeMLV in <span class="html-italic">A. mellifera</span> pupae (light grey), adults (medium grey) and <span class="html-italic">V. destructor</span> (dark grey) collected during Spring, Summer and Autumn of 2002 from 360 colonies in 36 French apiaries. The data are analyzed at the apiary level, and at the individual colony level for all seasons (adult samples only) and for all three sample types (autumn samples only); (<b>B</b>) Distribution of the number of infected colonies per apiary throughout the season, as determined from the adult colony samples.</p> Full article ">Figure 3 Cont.
<p>(<b>A</b>) Prevalence of BeeMLV in <span class="html-italic">A. mellifera</span> pupae (light grey), adults (medium grey) and <span class="html-italic">V. destructor</span> (dark grey) collected during Spring, Summer and Autumn of 2002 from 360 colonies in 36 French apiaries. The data are analyzed at the apiary level, and at the individual colony level for all seasons (adult samples only) and for all three sample types (autumn samples only); (<b>B</b>) Distribution of the number of infected colonies per apiary throughout the season, as determined from the adult colony samples.</p> Full article ">Figure 4
<p>Log<sub>10</sub>[BeeMLV] titers of genomic (g) and sub-genomic plus genomic (sg + g) BeeMLV RNA in pupae (Pu), adults (Ad) and varroa (V) samples collected in Autumn 2002 from four apiaries in Western (#208; blue), Northern (#28; green) Eastern (#802; orange) and Southern (#878; red) France. Values represent the mean and standard deviation of two independent assays for only genomic RNA, and two assays for genomic-plus-subgenomic RNA (located 5′ and 3′ respectively of the putative start of the sub-genomic RNA; <a href="#viruses-07-02789-f001" class="html-fig">Figure 1</a>), run on the pooled cDNA from 10 colonies in each apiary, for each of the sample types. The faded area represents the limit of detection. Significant differences between the genomic and genomic-plus-subgenomic titers, as determined by <span class="html-italic">t</span>-tests, are marked by an asterisk (*).</p> Full article ">
115 KiB  
Review
Targeting CTCF to Control Virus Gene Expression: A Common Theme amongst Diverse DNA Viruses
by Ieisha Pentland and Joanna L. Parish
Viruses 2015, 7(7), 3574-3585; https://doi.org/10.3390/v7072791 - 6 Jul 2015
Cited by 29 | Viewed by 6471
Abstract
All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression [...] Read more.
All viruses target host cell factors for successful life cycle completion. Transcriptional control of DNA viruses by host cell factors is important in the temporal and spatial regulation of virus gene expression. Many of these factors are recruited to enhance virus gene expression and thereby increase virus production, but host cell factors can also restrict virus gene expression and productivity of infection. CCCTC binding factor (CTCF) is a host cell DNA binding protein important for the regulation of genomic chromatin boundaries, transcriptional control and enhancer element usage. CTCF also functions in RNA polymerase II regulation and in doing so can influence co-transcriptional splicing events. Several DNA viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human papillomavirus (HPV) utilize CTCF to control virus gene expression and many studies have highlighted a role for CTCF in the persistence of these diverse oncogenic viruses. CTCF can both enhance and repress virus gene expression and in some cases CTCF increases the complexity of alternatively spliced transcripts. This review article will discuss the function of CTCF in the life cycle of DNA viruses in the context of known host cell CTCF functions. Full article
(This article belongs to the Special Issue Tumour Viruses)
740 KiB  
Article
Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis
by Jose L. Nieto-Torres, Carmina Verdiá-Báguena, Carlos Castaño-Rodriguez, Vicente M. Aguilella and Luis Enjuanes
Viruses 2015, 7(7), 3552-3573; https://doi.org/10.3390/v7072786 - 3 Jul 2015
Cited by 65 | Viewed by 11414
Abstract
Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion [...] Read more.
Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. Full article
(This article belongs to the Special Issue Viroporins)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Ion channels formed by viroporins. Left depiction represents a channel exclusively formed by protein monomers (blue cylinders) inserted on a lipid membrane. Schematic on the right shows a protein-lipid pore. In this latter case, the lipid head groups (cyan circles) are oriented towards the channel pore, modulating ion conductance and selectivity.</p> Full article ">Figure 2
<p>Functional involvement of lipid head groups in the protein-lipid pore formed by SARS-CoV E protein. Depictions represent E protein channels inserted in non-charged membranes (left) or negatively-charged membranes (right), under low solute concentrations and neutral pH. In these circumstances, each E protein monomer presents two negative charges provided by glutamic acid residues, and a positive charge conferred by an arginine. When reconstituted in fully or partially negatively-charged membranes, lipid head groups provide additional negative charges to the pore, which makes E protein channel more selective for cations and more conductive.</p> Full article ">Figure 3
<p>Pathways stimulated by viroporin ion channel activity leading to virus production. Viral-membrane embedded viroporins (red ellipses) transport H<sup>+</sup> inside the endocytosed virion. This causes structural changes in fusion and matrix proteins facilitating the uncoating of viral ribonucleoproteins (<b>1</b>); Viroporin-mediated ions leak from intracellular organelles such as the endoplasmic reticulum (ER) or the Golgi apparatus towards the cytoplasm causes a blockade of vesicle transport and/or hijacking of autophagic membranes. These processes finally result in the accumulation of membranous structures that will serve as platforms for viral replication and morphogenesis. Blue, red and green structures show the viral replicase (<b>2</b>); In addition, equilibration of Golgi and secretory pathway organelles’ pH protect both viral proteins involved in entry (blue structures and green ellipses) and newly formed virions that can be sensitive to acidic environments (<b>3</b>); Viroporins (red and blue ellipses), locate in the budding neck of some enveloped viruses. These proteins may interact and oligomerize, rearranging the formation of channels, which additionally could facilitate virion scission (<b>4</b>).</p> Full article ">Figure 4
<p>Pathways stimulated by viroporin ion channel activity leading to pathology. Molecular patterns associated with viral infections are recognized by cellular sensors (signal 1), which activate the transcription and translation of the NLRP3 inflammasome components (NLRP3, ASC and procaspase-1) and the inactive pro-IL-1β. Viroporins inserted in the intracellular organelles, such as the endoplasmic reticulum (ER) or the Golgi apparatus, favor the leak of Ca<sup>2+</sup> and H<sup>+</sup> ions that move following their electrochemical gradient into cell cytoplasm. This ionic imbalance (signal 2) induces the assembly of the inflammasome complex, which triggers the maturation of pro-IL-1β into IL-1β through the action of caspase-1. Secreted IL-1β mediates a potent pro-inflammatory response that can be deleterious for the cell and the organism, when overstimulated. In addition, alteration of ionic milieus in intracellular compartments comes along with a protein transport delay or blockage. This results in a decrease of the levels of MHC-I molecules (blue rectangles) at the plasma membrane, preventing the infected cell to be recognized by the immune system. Protein transport blockage also diminishes the levels and activity in the cell surface of ion channels and transporters, crucial in the resolution of edema accumulation. Epithelial sodium channels (green structure) and Na<sup>+</sup>/K<sup>+</sup> ATPase (purple rectangles) impairment have been related to the worsening of viral respiratory diseases such as those caused by SARS-CoV, IAV or RSV.</p> Full article ">
906 KiB  
Review
The Human Papillomavirus E6 PDZ Binding Motif: From Life Cycle to Malignancy
by Ketaki Ganti, Justyna Broniarczyk, Wiem Manoubi, Paola Massimi, Suruchi Mittal, David Pim, Anita Szalmas, Jayashree Thatte, Miranda Thomas, Vjekoslav Tomaić and Lawrence Banks
Viruses 2015, 7(7), 3530-3551; https://doi.org/10.3390/v7072785 - 2 Jul 2015
Cited by 105 | Viewed by 11834
Abstract
Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. [...] Read more.
Cancer-causing HPV E6 oncoproteins are characterized by the presence of a PDZ binding motif (PBM) at their extreme carboxy terminus. It was long thought that this region of E6 had a sole function to confer interaction with a defined set of cellular substrates. However, more recent studies have shown that the E6 PBM has a complex pattern of regulation, whereby phosphorylation within the PBM can regulate interaction with two classes of cellular proteins: those containing PDZ domains and the members of the 14-3-3 family of proteins. In this review, we explore the roles that the PBM and its ligands play in the virus life cycle, and subsequently how these can inadvertently contribute towards the development of malignancy. We also explore how subtle alterations in cellular signal transduction pathways might result in aberrant E6 phosphorylation, which in turn might contribute towards disease progression. Full article
(This article belongs to the Special Issue Tumour Viruses)
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Figure 1

Figure 1
<p>Papillomavirus oncoproteins show diversity in the PBMs and their kinase recognition sequences. The multiple sequence alignment of various HPV E6 proteins from different HPV types and the MmPV E7 using the Clustal X color scheme for the ClustalW sequence alignment program [<a href="#B28-viruses-07-02785" class="html-bibr">28</a>] show variation in the sequences of their C-terminus, which includes the PBM in the high risk HPV E6 proteins as well as the MmPV1 E7 protein, which is absent in the low risk HPV E6 types. The 4 boxed amino acids at the extreme C-terminus designate the canonical PBM. Whilst residues at p0 and -2 form the basis of PDZ recognition, the residues marked (*) indicate the amino acids that have been shown to be important for the specificity of E6 interaction with Dlg, while residues marked (+) designate the amino acids crucial for MAGI-1 specificity. The consensus sequences for AKT and PKA recognition of the E6 proteins is also shown.</p> Full article ">Figure 2
<p>The role of the E6 PBM in the HPV life cycle and malignancy. The figure shows the productive life cycle of the virus after infection of the epithelium with coordinate expression of the different viral gene products during epithelial differentiation, ultimately resulting in the production of new infectious virus particles. The E6 PBM function, most likely through PDZ targeting, is required for expansion of replication competent cells and for maintenance of the viral episomal DNA. During differentiation and viral DNA amplification in the G2M like phase of the cell cycle, E6 will most likely be phosphorylated within the PBM, which could confer interaction with 14-3-3 proteins. Following a persistent infection of up to 20 years, the progression of HPV induced malignancy can occur. The role of the E6 PBM in this stage is unknown, but PDZ targeting might contribute to loss of cell polarity regulators and drive proliferation and invasion. Phosphorylation of the E6 PBM might be a means of negatively regulating this activity of E6.</p> Full article ">Figure 3
<p>Papillomavirus oncoprotein targeting of cell polarity proteins. The cartoon depicts the various proteins comprising the three major complexes that regulate cell polarity: apical is defined by the Crumbs (CRBS) complex, subapical by the Par complex and basolateral by the Scrib complex. These complexes interact through a series of mutually antagonistic interactions ensuring correct spatial distribution and levels of expression of the individual components. Note the propensity of HPV E6 and MmPV E7 to target diverse components of this cell polarity control network, thereby perturbing their levels of expression or subcellular distribution.</p> Full article ">
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