A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization
<p>Scheme of SARS2 genome and the SARS2 replicon DNA construct. (<b>A</b>). The full-length of SARS2 genome from the Wuhan-Hu-1 isolate (GenBank accession No. NC_045512.2) encodes 5′ untranslated region (UTR), nonstructural proteins NSP1-16, structural proteins S, E, M, and N, accessory proteins ORF3-10, and 3′UTR. (<b>B</b>). Several genetic elements were included in the recombinant replicon DNA construct for various purposes. These include HIV long terminal repeat (LTR) promoter, T7 promoter, hammerhead virus ribozyme (HHV Rz) at the 5′ end, porcine teschovirus-1 self-cleaving peptide 2A (P2A) between NSP1 aa1-183 and firefly luciferase, encephalomyocarditis virus internal ribosome entry site (IRES) before NSP2-16, green fluorescence protein-blasticidine (GFP::Bsr) in place of S/E/M, and hepatitis delta virus ribozyme (HDV Rz) and bovine growth hormone polyadenylation signal (BGH pA) at the 3′ end.</p> "> Figure 2
<p>Construction of the SARS2 replicon DNA. (<b>A</b>) The full-length of the recombinant SARS2 replicon DNA (27,952 bp) was divided into and synthesized in 5 fragments (F1/F6 and F2-F5) in the backbone of pMX backbone vector (for F2-5) or pMK backbone vector (for F1/6) with approximate nucleotide sequences for BsaI or SalI restriction sites at both 5′ and 3′ end. (<b>B</b>) Fragments F2-5 in pMX were ligated to create pMX.F2-5 construct using a Golden Gate Assembly kit. (<b>C</b>) pMXF2-5 and pMKF1/6 were digested with SalI and ligated to create the full-length recombinant non-infectious SARS2 replicon DNA construct using a homology recombination-based Gibson Assembly kit.</p> "> Figure 3
<p>Recombinant SARS2 replicon DNA and its transcribed replicon RNA. (<b>A</b>). The intermediate product pMXF2-5 DNA from the Golden Gate Assembly and pMK.F1/6 DNA were confirmed using 0.5% agarose gel electrophoresis. (<b>B</b>). The SARS replicon DNA was obtained from pMX.F2-5 and pMK.F1/6 using a Golden Gate Assembly kit and confirmed using 0.5% agarose gel electrophoresis, marked by an arrow. (<b>C</b>). The full-length recombinant SARS2 replicon DNA was confirmed by PCR using primer pairs spanning specific junctions between two adjacent DNA fragments. (<b>D</b>). SARS2 DNA replicon was used to synthesize SARS2 RNA replicon using an in vitro T7 transcription kit, and the RNA replicon was confirmed using denatured agarose electrophoresis (0.7%), marked by an arrow. Stand’s: DNA size markers.</p> "> Figure 4
<p>Luciferase reporter gene expression from recombinant SARS2 replicon DNA in response to HIV Tat expression (<b>A</b>,<b>B</b>). The 293T were plated at a density of 1.5 × 10<sup>5</sup> cells/well in a 24-well plate, transfected with 0.4 μg SARS2 replicon DNA and an increasing amount of pc3.Tat, cultured for 24 h, and harvested for the luciferase activity assay (<b>A</b>), or transfected with 0.4 μg SARS2 replicon DNA and 0.12 μg pc3.Tat, cultured for different lengths of time, and harvested for the luciferase activity assay (<b>B</b>,<b>C</b>). Vero E6 were transfected with 0.4 μg SARS2 replicon DNA and 0.12 μg pc3.Tat, cultured for different lengths of time, and harvested for the luciferase activity assay. pcDNA3 was used to equalize the total amount of DNA among all transfections. The data were Mean ± SEM and representative of at least three independent experiments. All differences were highly significant compared to Tat (0 μg) (<b>A</b>), and compared to Time (0 h), except Time (6 h) and between Replicon DNA and Replicon DNA + Tat (<b>B</b>,<b>C</b>).</p> "> Figure 5
<p>Luciferase reporter gene expression from SARS2 replicon RNA in response to HIV Tat expression. The 293T (<b>A</b>) or Vero E6 (<b>B</b>) were plated at a density of 1.5 × 10<sup>5</sup> cells/well for 293T and 1.5 × 10<sup>5</sup> cells/well for Vero E6 in a 24-well plate, transfected with 0.3 μg SARS2 replicon RNA and 0.1 μg pc3.Tat, cultured for different lengths of time, and harvested for the luciferase activity assay. pcDNA3 was used to equalize the total amount of DNA among all transfections. The data were Mean ± SEM and representative of at least three independent experiments. All differences were highly significant compared to Time (0 h) and insignificant between Replicon RNA and Replicon RNA + Tat.</p> "> Figure 6
<p>Expression of the GFP reporter gene and SARS2 N (<b>A</b>,<b>B</b>). The 293T were plated at a density of 4 × 10<sup>6</sup> cells/plate in a 10 cm plate, transfected with 10 μg SARS2 replicon DNA and 3.3 μg pc3.Tat, cultured for different lengths of time, and harvested for Western blotting and direct imaging of the GFP signal on the blots at 488 nM (<b>A</b>), or for Western blotting against an anti-SARS2 N antibody (<b>B</b>). Untx: 293T were only transfected with pcDNA3. (<b>C</b>). The 293T were at a density of 4 × 10<sup>6</sup> cells/plate in a 10 cm plate, transfected with 7.5 μg SARS2 replicon RNA, cultured for different lengths of time, and harvested for Western blotting against an anti-SARS2 N antibody. Western blotting against an anti-β-actin antibody was included as the equal loading control. The data were representative of at least three independent experiments.</p> "> Figure 7
<p>Expression of positive/negative-stranded genomic RNA (gRNA) and N subgenomic RNA (sgRNA) from the SARS2 replicon and its response to Remdesivir. (<b>A</b>) Different RT primers (N3′ and LRS-L) in combination with different PCR primers (N5′/N3′ and TSR-L/N3′) were designed to distinguish positive-stranded from negative-stranded gRNA and N sgRNA. RT with N3′, followed by PCR with N5′/N3′ and TSR-L/N3′ represented positive-stranded gRNA and N sgRNA, respectively. RT with TSR-L, followed by PCR with N5′/N3′ and TSR-L/N3′, represented negative-stranded gRNA and N sgRNA, respectively. (<b>B</b>) The 293T were plated at a density of 6.5 × 10<sup>5</sup> cells/well in a 6-well plate, treated with 0, 5, or 10 μM Remdesivir for 1 h, transfected with 1.5 μg SARS2 replicon DNA and 0.5 μg pcDNA3, 1.5 μg SARS2 DNA and 0.5 μg pc3.Tat, or 1.2 μg SARS2 replicon RNA, cultured in the presence of Remdesivir for 24 h, and harvested for RNA isolation. RT was performed using N3′ or TRS-L5′ as the primer and 0.5 μg RNA in a 25 μL reaction. An aliquot RT reaction (2 μL from N3′ RT reaction; 2 μL from TSR-L5′ RT reaction) was used as the template for PCR, with indicated primer pairs. The PCR products were analyzed on 1% agarose gel electrophoresis. RT was performed using 0.1 μg RNA. RT with oligo d(T)<sub>23</sub> as the RT primer and PCR with β-actin-specific primers were performed and included as the equal loading control. Stand’s: DNA size markers. Ctrl: untransfected cells. The data were representative of at least three independent experiments.</p> "> Figure 8
<p>Effects of Remdesivir on gene expression from the SARS2 replicon DNA and RNA (<b>A</b>,<b>B</b>). The 293T were at a density of 4 × 10<sup>6</sup> cells/plate in a 10 cm plate, treated with Remdesivir for 1 h, transfected with 10 μg SARS2 replicon DNA, cultured in the presence of Remdesivir for 24 h, and harvested for the luciferase activity assay (<b>A</b>), or for Western blotting against an anti-SARS2 N antibody or anti-β-actin antibody, or by direct imaging of the GFP signal at 488 nm (<b>B</b>). (<b>C</b>). The 293T were at a density of 1.5 × 10<sup>5</sup> cells/well in a 24-well plate, treated with 10 μM Remdesivir for 1 h, transfected with 0.4 μg SARS2 replicon DNA and 0.12 μg pcDNA3, 0.4 μg SARS2 DNA and 0.12 μg pc3.Tat, or 0.3 μg SARS2 replicon RNA and 0.1 μg yeast tRNA, cultured in the presence of Remdesivir for 24 h, and harvested for the luciferase activity assay. The controls for Remdesivir treatment were DMSO, the solvent for Remdesivir. The data were Mean ± SEM and representative of at least three independent experiments (<b>A</b>,<b>C</b>) and representative of at least three independent experiments (<b>B</b>). All differences were highly significant compared to Remdesivir (0 μM) (<b>A</b>) and between ± Remdesivir (<b>C</b>).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Transfection, and Remdesivir Treatment
2.2. Synthesis of Replicon Fragments and Construction of Recombinant Non-Infectious SARS2 Replicon DNA
2.3. Purification of the SARS2 Replicon Plasmid DNA pMK.F1–6 and In Vitro RNA Transcription
2.4. Luciferase Reporter Gene Assay
2.5. Western Blotting
2.6. Semi-Quantitative RT-PCR Determination of (+) and (−) Strand SARS2 Replicon Genomic RNA (gRNA) or N Subgenomic RNA (sgRNA)
2.7. Data Analysis
3. Results
3.1. Design and Construction of the LTR/T7 Dual-Promoter-Driven and GFP/fLuc Dual-Reporter-Expressing SARS2 Replicon
3.2. Expression of the Reporter Genes and SARS2 N Gene from the LTR/T7 Dual-Promoter-Driven and GFP/fLuc Dual-Reporter-Expressing SARS2 Replicon
3.3. RNA Transcription and Replication from the LTR/T7 Dual-Promoter-Driven and GFP/fLuc Dual-Reporter-Expressing SARS2 Replicon and its Inhibition by Remdesivir
3.4. Inhibition of Gene Expression from the LTR/T7 Dual-Promoter-Driven and GFP/fLuc Dual-Reporter-Expressing SARS2 Replicon by Remdesivir
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Zhang, G.; Zhang, J.; Wang, B.; Zhu, X.; Wang, Q.; Qiu, S. Analysis of clinical characteristics and laboratory findings of 95 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A retrospective analysis. Respir. Res. 2020, 21, 74. [Google Scholar] [CrossRef] [PubMed]
- Tan, W.; Zhao, X.; Ma, X.; Wang, W.; Niu, P.; Xu, W.; Gao, G.F.; Wu, G. A Novel Coronavirus Genome Identified in a Cluster of Pneumonia Cases—Wuhan, China 2019–2020. China CDC Wkly. 2020, 2, 61–62. [Google Scholar] [CrossRef] [PubMed]
- Meng, H.; Xiong, R.; He, R.; Lin, W.; Hao, B.; Zhang, L.; Lu, Z.; Shen, X.; Fan, T.; Jiang, W.; et al. CT imaging and clinical course of asymptomatic cases with COVID-19 pneumonia at admission in Wuhan, China. J. Infect. 2020, 81, e33–e39. [Google Scholar] [CrossRef] [PubMed]
- Chen, N.; Zhou, M.; Dong, X.; Qu, J.; Gong, F.; Han, Y.; Qiu, Y.; Wang, J.; Liu, Y.; Wei, Y.; et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet 2020, 395, 507–513. [Google Scholar] [CrossRef] [Green Version]
- World Health Organization Coronavirus (COVID-19) Dashboard. 2022. Available online: http://COVID19.WHO.int (accessed on 19 April 2022).
- Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, 270–273. [Google Scholar] [CrossRef] [Green Version]
- Pu, R.; Liu, S.; Ren, X.; Shi, D.; Ba, Y.; Huo, Y.; Zhang, W.; Ma, L.; Liu, Y.; Yang, Y.; et al. The screening value of RT-LAMP and RT-PCR in the diagnosis of COVID-19: Systematic review and meta-analysis. J. Virol. Methods 2022, 300, 114392. [Google Scholar] [CrossRef]
- Ionescu, M.A. COVID-19 skin lesions are rarely positive at RT-PCR test: The macrophage activation with vascular impact and SARS-CoV-2-induced cytokine storm. Int. J. Dermatol. 2022, 61, 3–6. [Google Scholar] [CrossRef]
- Szabo, G.T.; Mahiny, A.J.; Vlatkovic, I. COVID-19 mRNA vaccines: Platforms and current developments. Mol. Ther. 2022, 30, 1850–1868. [Google Scholar] [CrossRef]
- Rahman, M.M.; Masum, M.H.U.; Wajed, S.; Talukder, A. A comprehensive review on COVID-19 vaccines: Development, effectiveness, adverse effects, distribution and challenges. Virusdisease 2022, 33, 1–22. [Google Scholar] [CrossRef]
- Jensen, A.; Stromme, M.; Moyassari, S.; Chadha, A.S.; Tartaglia, M.C.; Szoeke, C.; Ferretti, M.T. COVID-19 vaccines: Considering sex differences in efficacy and safety. Contemp. Clin. Trials 2022, 115, 106700. [Google Scholar] [CrossRef]
- Huang, Z.; Su, Y.; Zhang, T.; Xia, N. A review of the safety and efficacy of current COVID-19 vaccines. Front. Med. 2022, 16, 39–55. [Google Scholar] [CrossRef]
- Abbott, T.R.; Dhamdhere, G.; Liu, Y.; Lin, X.; Goudy, L.; Zeng, L.; Chemparathy, A.; Chmura, S.; Heaton, N.S.; Debs, R.; et al. Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell 2020, 181, 865–876.e812. [Google Scholar] [CrossRef]
- Fareh, M.; Zhao, W.; Hu, W.; Casan, J.M.L.; Kumar, A.; Symons, J.; Zerbato, J.M.; Fong, D.; Voskoboinik, I.; Ekert, P.G.; et al. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. Nat. Commun. 2021, 12, 4270. [Google Scholar] [CrossRef]
- Vitiello, A.; Ferrara, F. Association and pharmacological synergism of the triple drug therapy baricitinib/remdesivir/rhACE2 for the management of COVID-19 infection. Naunyn Schmiedeberg’s Arch. Pharmacol. 2022, 395, 99–104. [Google Scholar] [CrossRef]
- Pagliano, P.; Sellitto, C.; Scarpati, G.; Ascione, T.; Conti, V.; Franci, G.; Piazza, O.; Filippelli, A. An overview of the preclinical discovery and development of remdesivir for the treatment of coronavirus disease 2019 (COVID-19). Expert Opin. Drug Discov. 2021, 17, 9–18. [Google Scholar] [CrossRef]
- Kaka, A.S.; MacDonald, R.; Linskens, E.J.; Langsetmo, L.; Vela, K.; Duan-Porter, W.; Wilt, T.J. Major Update 2: Remdesivir for Adults With COVID-19: A Living Systematic Review and Meta-analysis for the American College of Physicians Practice Points. Ann. Intern. Med. 2022. [Google Scholar] [CrossRef]
- Angamo, M.T.; Mohammed, M.A.; Peterson, G.M. Efficacy and safety of remdesivir in hospitalised COVID-19 patients: A systematic review and meta-analysis. Infection 2022, 50, 27–41. [Google Scholar] [CrossRef]
- Pourkarim, F.; Pourtaghi-Anvarian, S.; Rezaee, H. Molnupiravir: A new candidate for COVID-19 treatment. Pharmacol. Res. Perspect. 2022, 10, e00909. [Google Scholar] [CrossRef]
- Khiali, S.; Khani, E.; Rouy, S.B.; Entezari-Maleki, T. Comprehensive review on molnupiravir in COVID-19: A novel promising antiviral to combat the pandemic. Future Microbiol. 2022, 17, 377–391. [Google Scholar] [CrossRef]
- Thye, A.Y.; Law, J.W.; Tan, L.T.; Pusparajah, P.; Ser, H.L.; Thurairajasingam, S.; Letchumanan, V.; Lee, L.H. Psychological Symptoms in COVID-19 Patients: Insights into Pathophysiology and Risk Factors of Long COVID-19. Biology 2022, 11, 61. [Google Scholar] [CrossRef]
- Thallapureddy, K.; Thallapureddy, K.; Zerda, E.; Suresh, N.; Kamat, D.; Rajasekaran, K.; Moreira, A. Long-Term Complications of COVID-19 Infection in Adolescents and Children. Curr. Pediatr. Rep. 2022, 10, 11–17. [Google Scholar] [CrossRef]
- Mehandru, S.; Merad, M. Pathological sequelae of long-haul COVID. Nat. Immunol. 2022, 23, 194–202. [Google Scholar] [CrossRef]
- Joshee, S.; Vatti, N.; Chang, C. Long-Term Effects of COVID-19. Mayo Clin. Proc. 2022, 97, 579–599. [Google Scholar] [CrossRef]
- Han, Q.; Zheng, B.; Daines, L.; Sheikh, A. Long-Term Sequelae of COVID-19: A Systematic Review and Meta-Analysis of One-Year Follow-Up Studies on Post-COVID Symptoms. Pathogens 2022, 11, 269. [Google Scholar] [CrossRef] [PubMed]
- Desai, A.D.; Lavelle, M.; Boursiquot, B.C.; Wan, E.Y. Long-term complications of COVID-19. Am. J. Physiol. Cell Physiol. 2022, 322, C1–C11. [Google Scholar] [CrossRef] [PubMed]
- Kim, D.; Lee, J.Y.; Yang, J.S.; Kim, J.W.; Kim, V.N.; Chang, H. The Architecture of SARS-CoV-2 Transcriptome. Cell 2020, 181, 914–921.e910. [Google Scholar] [CrossRef] [PubMed]
- Snijder, E.J.; Decroly, E.; Ziebuhr, J. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing. Adv. Virus Res. 2016, 96, 59–126. [Google Scholar] [CrossRef] [PubMed]
- Sola, I.; Almazan, F.; Zuniga, S.; Enjuanes, L. Continuous and Discontinuous RNA Synthesis in Coronaviruses. Annu. Rev. Virol. 2015, 2, 265–288. [Google Scholar] [CrossRef] [Green Version]
- Kaplan, G.; Racaniello, V.R. Construction and characterization of poliovirus subgenomic replicons. J. Virol. 1988, 62, 1687–1696. [Google Scholar] [CrossRef] [Green Version]
- Thumfart, J.O.; Meyers, G. Feline calicivirus: Recovery of wild-type and recombinant viruses after transfection of cRNA or cDNA constructs. J. Virol. 2002, 76, 6398–6407. [Google Scholar] [CrossRef] [Green Version]
- Liljestrom, P.; Garoff, H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology 1991, 9, 1356–1361. [Google Scholar] [CrossRef]
- Khromykh, A.A.; Westaway, E.G. Subgenomic replicons of the flavivirus Kunjin: Construction and applications. J. Virol. 1997, 71, 1497–1505. [Google Scholar] [CrossRef] [Green Version]
- Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theilmann, L.; Bartenschlager, R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999, 285, 110–113. [Google Scholar] [CrossRef] [Green Version]
- Behrens, S.E.; Grassmann, C.W.; Thiel, H.J.; Meyers, G.; Tautz, N. Characterization of an autonomous subgenomic pestivirus RNA replicon. J. Virol. 1998, 72, 2364–2372. [Google Scholar] [CrossRef] [Green Version]
- Pang, X.; Zhang, M.; Dayton, A.I. Development of Dengue virus type 2 replicons capable of prolonged expression in host cells. BMC Microbiol. 2001, 1, 18. [Google Scholar] [CrossRef] [Green Version]
- Shi, P.Y.; Tilgner, M.; Lo, M.K. Construction and characterization of subgenomic replicons of New York strain of West Nile virus. Virology 2002, 296, 219–233. [Google Scholar] [CrossRef]
- Hertzig, T.; Scandella, E.; Schelle, B.; Ziebuhr, J.; Siddell, S.G.; Ludewig, B.; Thiel, V. Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA. J. Gen. Virol. 2004, 85, 1717–1725. [Google Scholar] [CrossRef]
- Ge, F.; Luo, Y.; Liew, P.X.; Hung, E. Derivation of a novel SARS-coronavirus replicon cell line and its application for anti-SARS drug screening. Virology 2007, 360, 150–158. [Google Scholar] [CrossRef]
- Ge, F.; Xiong, S.; Lin, F.S.; Zhang, Z.P.; Zhang, X.E. High-throughput assay using a GFP-expressing replicon for SARS-CoV drug discovery. Antivir. Res. 2008, 80, 107–113. [Google Scholar] [CrossRef]
- Almazan, F.; Galan, C.; Enjuanes, L. The nucleoprotein is required for efficient coronavirus genome replication. J. Virol. 2004, 78, 12683–12688. [Google Scholar] [CrossRef] [Green Version]
- Wang, J.M.; Wang, L.F.; Shi, Z.L. Construction of a non-infectious SARS coronavirus replicon for application in drug screening and analysis of viral protein function. Biochem. Biophys. Res. Commun. 2008, 374, 138–142. [Google Scholar] [CrossRef] [PubMed]
- Bartenschlager, R. Hepatitis C virus replicons: Potential role for drug development. Nat. Rev. Drug Discov. 2002, 1, 911–916. [Google Scholar] [CrossRef]
- Randall, G.; Rice, C.M. Hepatitis C virus cell culture replication systems: Their potential use for the development of antiviral therapies. Curr. Opin. Infect. Dis. 2001, 14, 743–747. [Google Scholar] [CrossRef] [PubMed]
- Berkhout, B.; Gatignol, A.; Rabson, A.B.; Jeang, K.T. TAR-independent activation of the HIV-1 LTR: Evidence that tat requires specific regions of the promoter. Cell 1990, 62, 757–767. [Google Scholar] [CrossRef]
- Jakobovits, A.; Rosenthal, A.; Capon, D.J. Trans-activation of HIV-1 LTR-directed gene expression by tat requires protein kinase C. EMBO J. 1990, 9, 1165–1170. [Google Scholar] [CrossRef] [PubMed]
- Jeang, K.T.; Shank, P.R.; Kumar, A. Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis. Proc. Natl. Acad. Sci. USA 1988, 85, 8291–8295. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Selby, M.J.; Bain, E.S.; Luciw, P.A.; Peterlin, B.M. Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. Genes Dev. 1989, 3, 547–558. [Google Scholar] [CrossRef] [Green Version]
- Chang, J.J.; Rawlinson, D.; Pitt, M.E.; Taiaroa, G.; Gleeson, J.; Zhou, C.; Mordant, F.L.; De Paoli-Iseppi, R.; Caly, L.; Purcell, D.F.J.; et al. Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection. Cell Rep. 2021, 35, 109108. [Google Scholar] [CrossRef]
- Davidson, A.D.; Williamson, M.K.; Lewis, S.; Shoemark, D.; Carroll, M.W.; Heesom, K.J.; Zambon, M.; Ellis, J.; Lewis, P.A.; Hiscox, J.A.; et al. Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein. Genome Med. 2020, 12, 68. [Google Scholar] [CrossRef]
- V’Kovski, P.; Kratzel, A.; Steiner, S.; Stalder, H.; Thiel, V. Coronavirus biology and replication: Implications for SARS-CoV-2. Nat. Rev. Microbiol. 2021, 19, 155–170. [Google Scholar] [CrossRef]
- Wang, D.; Jiang, A.; Feng, J.; Li, G.; Guo, D.; Sajid, M.; Wu, K.; Zhang, Q.; Ponty, Y.; Will, S.; et al. The SARS-CoV-2 subgenome landscape and its novel regulatory features. Mol. Cell 2021, 81, 2135–2147.e2135. [Google Scholar] [CrossRef]
- Yang, Y.; Yan, W.; Hall, A.B.; Jiang, X. Characterizing Transcriptional Regulatory Sequences in Coronaviruses and Their Role in Recombination. Mol. Biol. Evol. 2021, 38, 1241–1248. [Google Scholar] [CrossRef]
- Beigel, J.H.; Tomashek, K.M.; Dodd, L.E.; Mehta, A.K.; Zingman, B.S.; Kalil, A.C.; Hohmann, E.; Chu, H.Y.; Luetkemeyer, A.; Kline, S.; et al. Remdesivir for the Treatment of Covid-19—Final Report. N. Engl. J. Med. 2020, 383, 1813–1826. [Google Scholar] [CrossRef]
- Gordon, C.J.; Tchesnokov, E.P.; Feng, J.Y.; Porter, D.P.; Gotte, M. The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus. J. Biol. Chem. 2020, 295, 4773–4779. [Google Scholar] [CrossRef] [Green Version]
- Grein, J.; Ohmagari, N.; Shin, D.; Diaz, G.; Asperges, E.; Castagna, A.; Feldt, T.; Green, G.; Green, M.L.; Lescure, F.X.; et al. Compassionate Use of Remdesivir for Patients with Severe Covid-19. N. Engl. J. Med. 2020, 382, 2327–2336. [Google Scholar] [CrossRef]
- Kokic, G.; Hillen, H.S.; Tegunov, D.; Dienemann, C.; Seitz, F.; Schmitzova, J.; Farnung, L.; Siewert, A.; Hobartner, C.; Cramer, P. Mechanism of SARS-CoV-2 polymerase stalling by remdesivir. Nat. Commun. 2021, 12, 279. [Google Scholar] [CrossRef]
- Wang, Q.; Wu, J.; Wang, H.; Gao, Y.; Liu, Q.; Mu, A.; Ji, W.; Yan, L.; Zhu, Y.; Zhu, C.; et al. Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase. Cell 2020, 182, 417–428.e413. [Google Scholar] [CrossRef]
- Liu, S.; Chou, C.K.; Wu, W.W.; Luan, B.; Wang, T.T. Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen. J. Virol. 2022, 96. [Google Scholar] [CrossRef]
- Ricardo-Lax, I.; Luna, J.M.; Thao, T.T.N.; Le Pen, J.; Yu, Y.; Hoffmann, H.H.; Schneider, W.M.; Razooky, B.S.; Fernandez-Martinez, J.; Schmidt, F.; et al. Replication and single-cycle delivery of SARS-CoV-2 replicons. Science 2021, 374, 1099–1106. [Google Scholar] [CrossRef]
- Zhang, Q.Y.; Deng, C.L.; Liu, J.; Li, J.Q.; Zhang, H.Q.; Li, N.; Zhang, Y.N.; Li, X.D.; Zhang, B.; Xu, Y.; et al. SARS-CoV-2 replicon for high-throughput antiviral screening. J. Gen. Virol. 2021, 102, 001583. [Google Scholar] [CrossRef]
- Kotaki, T.; Xie, X.; Shi, P.Y.; Kameoka, M. A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation. Sci. Rep. 2021, 11, 2229. [Google Scholar] [CrossRef]
- He, X.; Quan, S.; Xu, M.; Rodriguez, S.; Goh, S.L.; Wei, J.; Fridman, A.; Koeplinger, K.A.; Carroll, S.S.; Grobler, J.A.; et al. Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing. Proc. Natl. Acad. Sci. USA 2021, 118, e2025866118. [Google Scholar] [CrossRef]
- Zhang, H.; Fischer, D.K.; Shuda, M.; Moore, P.S.; Gao, S.J.; Ambrose, Z.; Guo, H. Construction and characterization of two SARS-CoV-2 minigenome replicon systems. J. Med. Virol. 2022, 94, 2438–2452. [Google Scholar] [CrossRef]
- Nguyen, H.T.; Falzarano, D.; Gerdts, V.; Liu, Q. Construction of a Noninfectious SARS-CoV-2 Replicon for Antiviral-Drug Testing and Gene Function Studies. J. Virol. 2021, 95, e0068721. [Google Scholar] [CrossRef]
- Wang, B.; Zhang, C.; Lei, X.; Ren, L.; Zhao, Z.; Wang, J.; Huang, H. Construction of Non-infectious SARS-CoV-2 Replicons and Their Application in Drug Evaluation. Virol. Sin. 2021, 36, 890–900. [Google Scholar] [CrossRef]
- Luo, Y.; Yu, F.; Zhou, M.; Liu, Y.; Xia, B.; Zhang, X.; Liu, J.; Zhang, J.; Du, Y.; Li, R.; et al. Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors. mBio 2021, 12, e02754-20. [Google Scholar] [CrossRef]
- Tanaka, T.; Saito, A.; Suzuki, T.; Miyamoto, Y.; Takayama, K.; Okamoto, T.; Moriishi, K. Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening. Antivir. Res. 2022, 199, 105268. [Google Scholar] [CrossRef] [PubMed]
Feature | Abbreviation | Location | Size (bp) | Function |
---|---|---|---|---|
HIV-1 LTR | LTR | 1–713 | 713 | To facilitate expression of long RNA transcript when transfected with HIV Tat |
T7 promoter | T7 | 722–740 | 19 | To synthesize viral RNA by in vitro transcription |
Hammerhead virus ribozyme | HHV Rz | 740–799 | 59 | To produce the native 5′ end of SARS2 viral RNA genome |
SARS2 5′ untranslated region | 5′UTR | 800–1064 | 265 | To maintain the regulatory element for SARS2 replication |
SARS2 nonstructural protein 1 | NSP1 | 1065–1604 | 549 | To encode NSP1 |
Porcine teschovirus-1 self-cleaving peptide 2A | P2A | 1611–1671 | 66 | To cleave NSP1-fLuc fusion protein to ensure proper fLuc expression |
Firefly luciferase | fLuc | 1677–3329 | 1653 | To monitor translation from and replication of SARS2 RNA |
Encephalomarcarditis virus internal ribozyme entry site | IRES | 3330–3910 | 581 | To facilitate translation of the long transcript of SARS2 NSP2-16 |
SARS2 nonstrctural protein 2-16 | NSP2-16 | 3917–24,666 | 20750 | To encode nonstructural SARS proteins NSP2-16 for replication |
Transcriptional regulatory sequence 1 | TRS1 | 24,675–24,681 | 7 | To maintain the authentic regulatory element for GFP::Bsr expression |
GFP-blasticidin S resistance fusion protein | GFP::Bsr | 24,682–25,998 | 1317 | To select stable SARS2 replicon and monitor SARS2 replicon replication |
Transcriptional regulatory sequence 2 | TRS2 | 25,999–26,102 | 14 | To maintain the authentic regulatory element for SARS2 nucleocapsid expression |
SARS2 nucleocapsid protein | N | 26,013–27,272 | 1260 | To encode SARS2 nucleocapsid and monitor SARS2 replicon replication |
SARS2 ORF10-3′ untraslated region | ORF10-3′UTR | 27,373–27,642 | 370 | To maintain the regulatory element integrity for SARS2 replication |
Hepatitis delta virus ribozyme | HDV Rz | 27,643–27,721 | 79 | To produce the native 3′ end of SARS2 viral RNA genome |
Bovine growth hormone polyadenylation signal | BGH pA | 27,728–27,952 | 225 | To stabilize the RNA transcript |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Liu, Y.; Li, L.; Timani, K.A.; He, J.J. A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization. Viruses 2022, 14, 974. https://doi.org/10.3390/v14050974
Liu Y, Li L, Timani KA, He JJ. A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization. Viruses. 2022; 14(5):974. https://doi.org/10.3390/v14050974
Chicago/Turabian StyleLiu, Ying, Lu Li, Khalid A. Timani, and Johnny J. He. 2022. "A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization" Viruses 14, no. 5: 974. https://doi.org/10.3390/v14050974