Anti-Adenoviral Effect of Human Argonaute 2 Alone and in Combination with Artificial microRNAs
"> Figure 1
<p>Schematic representation of the adenoviral vectors used in this study. All vectors were based on the HAd5V-derived vectors pAd/PL-DEST ™ (ThermoFisher Scientific, Vienna, Austria) lacking the E1 and E3 genes. Expression cassettes were inserted into the deleted E1 region in antisense orientation with respect to the left inverted terminal repeat (ITR). The expression cassettes contain EGFP [<a href="#B34-cells-13-01117" class="html-bibr">34</a>] or AGO2 (this study) open reading frames, either alone or in conjunction with six tandemly repeated, either targeting (pTP-mi5) or non-targeting (NT), amiRNA hairpins incorporated into the 3′ UTR of the EGFP and AGO2 transcripts, respectively. Expression is driven by a CMV promoter (pCMV).</p> "> Figure 2
<p>AGO2 negatively affects the HAdV-5 life cycle. (<b>A</b>) Overexpression of AGO2 decreases the wt HAdV-5 genome copy numbers. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with 250 ng plasmid and infected with wt HAdV-5 at an MOI of 0.1, respectively. Concentrations of HAdV-5 genome copy numbers were measured at 96 h post-transfection/infection using an E3-probe qPCR. The data display means and standard deviations from a total of 3 independent experiments. Each independent experiment’s EGFP control preparation measurement was set as a reference at 100%. ** (<span class="html-italic">p</span> < 0.01). (<b>B</b>) Overexpression of AGO2 has no effect on the adenovirus mutant dl-sub720. Same as in (<b>A</b>), except that cells were infected with the HAdV-5 mutant dl-sub720 instead of wt HAdV-5. ns (not significant). (<b>C</b>) Compared to wt AGO2 mutants defective for miRNA binding, they differ in their ability to decrease wt HAdV-5 genome copy numbers. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with 250 ng plasmid expressing either AGO2 or mutants thereof and were infected with wt HAdV-5 at an MOI of 0.1. Concentrations of HAdV-5 genome copy numbers were measured at 96 h post-transfection/infection using an E3-probe qPCR. The data represent the means of 3 representative experiments, including standard deviations. The mean value for the AGO2 measurements was set as a reference at 100%. * (<span class="html-italic">p</span> < 0.05); ** (<span class="html-italic">p</span> < 0.01). (<b>D</b>) The inhibition of HAdV-5 replication by AGO2 is comparable in A549 and HeLa cells. 1.5 × 10<sup>4</sup> A549 or HeLa cells were transduced with AGO2-expressing rAdV vectors at an MOI of 100. 12 h after transduction wt HAdV-5 was added at an MOI of 0.1. Concentrations of HAdV-5 genome copy numbers were measured at 48 h post-infection using an E3-probe qPCR. The data were derived from a total of 3 representative experiments and display mean ± standard deviations. Each experiment’s EGFP control preparation measurement was set as a reference at 100%. * (<span class="html-italic">p</span> < 0.05); ** (<span class="html-italic">p</span> < 0.01); ns (not significant).</p> "> Figure 3
<p>Putative target sites of cellular miRNAs within HAdV-5 early mRNA transcripts Pol and pTP as per in silico TargetScan analysis. In addition to putative target sites of cellular miRNAs (red arrows), the target sites of the previously described siRNAs Pol-si2 (blue arrow) and pTP-si8 [<a href="#B42-cells-13-01117" class="html-bibr">42</a>] with their corresponding amiRNA pTPmi5 (green arrow) [<a href="#B34-cells-13-01117" class="html-bibr">34</a>] are indicated.</p> "> Figure 4
<p>Targeting of HAdV-5 DNA polymerase and pTP sequences by cellular miRNAs and their impact on viral replication. (<b>A</b>) Schematic representation of the dual-luciferase reporter vector system employed in this study. Reporter vectors contain sequences of the HAdV-5 DNA polymerase and pTP genes (Pol; pTP) inserted into the 3′UTR of a Renilla luciferase reporter gene (RLuc). MiRNAs capable of recognizing the respective target mRNAs are expected to knock down Renilla luciferase expression relative to the expression of a non-targeted firefly luciferase gene (FLuc) present on the same vector. (<b>B</b>) Targeting of the HAdV-5 DNA polymerase mRNA by miRNA mimics in reporter assays. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with a HAdV-5 DNA polymerase reporter vector carrying the DNA polymerase sequence inserted into the 3′UTR of a Renilla luciferase reporter gene and endogenous miRNA mimics, respectively. A non-targeting (NT) miRNA mimic was used as a control. Readout as per manufacturers’ instructions was conducted at 48 h post-transfection and relative light units (RLUs) for the Renilla luciferase reporter gene were normalized to those of the firefly luciferase reporter gene. Each experiment’s NT control preparation measurement was set as a reference at 100%. The data represent means ± standard deviation of 3 experiments. * (<span class="html-italic">p</span> < 0.05). (<b>C</b>) Targeting of the HAdV-5 pTP mRNA by miRNA mimics in reporter assays. Same as in (<b>B</b>) except that a reporter vector carrying the HAdV-5 pTP sequence was used. (<b>D</b>) Effect of the hsa-miR-7 mimic on HAdV-5 genome copy numbers. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with an hsa-miR-7 mimic at a concentration of 10 nM and infected with HAdV-5 at an MOI of 0.1. Concentrations of HAdV-5 genome copy numbers were measured at 48 h post-transfection/infection using an E3-probe qPCR. Data represent mean ± standard deviations of a representative experiment carried out in triplicate. The non-targeting (NT) control preparation measurement was set as a reference at 100%. ** (<span class="html-italic">p</span> < 0.01).</p> "> Figure 5
<p>Inhibition of miRNA function with miRNA inhibitors and enhancement of miRNA-mediated effects on HAdV-5 by overexpression of AGO2. (<b>A</b>) Inhibition of the action of miRNA mimics with miRNA inhibitors in reporter assays. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with (i) a HAdV-5 DNA polymerase reporter vector carrying the DNA polymerase sequence inserted into the 3′UTR of a Renilla luciferase reporter gene, (ii) miRNA mimics, and (iii) corresponding miRNA Power Inhibitors (PI). Readout as per manufacturers’ instructions was conducted at 48 h post-transfection. Data (relative light units; RLU) derives from a total of 3 experiments and displays means ± standard deviations. Each experiment’s non-targeting (NT) control preparation measurement was set as a reference at 100%. ** (<span class="html-italic">p</span> < 0.01). (<b>B</b>) Effect of miRNA inhibitors in the absence of miRNA mimics in reporter assays. Same as in (<b>A</b>) except that no miRNA mimics directed against the reporter transcript were employed. ** (<span class="html-italic">p</span> < 0.01). (<b>C</b>) Effect of miRNA mimics on HAdV-5 replication. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transfected with 5 nM miRNA mimics and transduced with an AGO2- or EGFP-expressing adenoviral vector at an MOI of 100. 24 h later, wt HAdV-5 was added at an MOI of 0.1. Concentrations of wt HAdV-5 genome copy numbers were measured at 48 h post-infection using an E3-probe qPCR. Data represent means ± standard deviations of a representative experiment carried out in triplicate. * (<span class="html-italic">p</span> < 0.05).</p> "> Figure 6
<p>Improved amiRNA-mediated inhibition of wt HAdV-5 replication upon co-transduction with an AGO2-expressing rAdV vector. (<b>A</b>) The potency of an amiRNA targeting the adenoviral pTP mRNA is significantly improved upon overexpression of AGO2. 1.5 × 10<sup>4</sup> cells were simultaneously transduced with (i) a rAdV expressing HAdV-5-targeting (pTP-mi5) or non-targeting (NT) amiRNAs and (ii) a rAdV expressing AGO2 or EGFP at an MOI of 250, before being exposed to wt HAdV-5 at an MOI of 1. Concentrations of wt HAdV-5 genome copy numbers were measured at 72 h post-infection using an E3-probe qPCR. Each experiment’s EGFP control preparation measurement was set as a reference at 100%. The inhibition by AGO2 alone in the absence of any targeting or non-targeting amiRNA is shown for comparison. Data were derived from a total of 3 representative experiments and display mean ± standard deviations. * (<span class="html-italic">p</span> < 0.05); ns (not significant). AGO2-mediated differences between 6xNT and 6xpTP-mi5 were significant in all instances. (<b>B</b>) Evaluation of the impact of adenoviral vector MOIs in relation to wt HAdV-5 MOIs on viral replication and vector mobilization. 1.5 × 10<sup>4</sup> HeLa cells were simultaneously transduced with rAdVs at MOIs as per the X-axis and infected with wt HAdV-5 as per the Y-axis. Concentrations of wt HAdV-5 and vector genome copy numbers were measured at 48 h post-infection using E3- and CMV promoter-specific qPCR probes, respectively.</p> "> Figure 7
<p>Improvement of rAdV amiRNA vector performance by inclusion of an AGO2 expression cassette. (<b>A</b>) In a prophylactic treatment scenario, 1.5 × 10<sup>4</sup> HeLa cells were transduced with vectors at an MOI of 100 24 h prior to infection with HAdV-5 at an MOI of 0.1. Concentrations of wt HAdV-5 infectious particles (TCID50/mL) were measured at timepoints 0 (D0), 48 h (D2), 96 h (D4), and 144 h (D6). Data were derived from a total of 3 experiments and display mean ± standard deviations. **** (<span class="html-italic">p</span> < 0.0001). Significances in green: 6xNT+EGFP vs. 6xpTP-mi5+EGFP; significances in red: 6xNT+AGO2 vs. 6xpTP-mi5+AGO2. (<b>B</b>) In a therapeutic treatment scenario 1.5 × 10<sup>4</sup> HeLa cells underwent concomitant transduction and infection with vectors at an MOI of 100 as well as HAdV-5 at an MOI of 0.1, respectively. Concentrations of wt HAdV-5 infectious particles (TCID50/mL) were measured at timepoints 0 (D0), 48 h (D2), 96 h (D4), and 144 h (D6). Data were derived from a total of 3 experiments and display mean ± standard deviations. **** (<span class="html-italic">p</span> < 0.0001); ** (<span class="html-italic">p</span> < 0.01); * (<span class="html-italic">p</span> < 0.05). Significances in green: 6xNT+EGFP vs. 6xpTP-mi5+EGFP; significances in red: 6xNT+AGO2 vs. 6xpTP-mi5+AGO2.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture, Virus Amplification, and Titer Determination
2.2. Plasmid and Adenoviral Vector Construction
2.3. Nucleic Acid Extraction
2.4. Dual-Luciferase Assays
2.5. Virus Inhibition Experiments
2.6. Determination of Adenovirus Genome Copy Numbers
2.7. Determination of Adenovirus Infectious Particle Numbers
2.8. Bioinformatic ANALYSIS
2.9. miRNA Mimics
2.10. Power Inhibitors
2.11. Western Blotting
2.12. Statistical Analysis
3. Results
3.1. Ectopic Expression of Human AGO2 Impairs the HAdV-5 Life Cycle
3.2. In Silico Analysis Identifies Putative, Cellular, and Anti-Adenoviral miRNAs
3.3. Human miRNA-7 Impairs the wt HAdV-5 Life Cycle
3.4. AGO2 Co-Expression Improves the Performance of Anti-Adenoviral miRNAs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No. | hsa-miR- | TargetScan Score | 5p [5′→3′] | 3p [5′→3′] | Target Site 5′ | Target Site 3′ | Target Site Characteristic |
---|---|---|---|---|---|---|---|
1 | let-7a-5p | −530 | UGAGGUAGUAGGUUGUAUAGUU | CUAUACAAUCUACUGUCUUUC | 4229 | 4251 | pTP+Pol 3′UTR |
let-7a-5p | −626 | 9120 | 9142 | pTP ORF | |||
let-7a-5p | −428 | 8612 | 8634 | pTP ORF | |||
2 | miR-100-5p | −698 | AACCCGUAGAUCCGAACUUGUG | CAAGCUUGUAUCUAUAGGUAUG | 6258 | 6280 | Pol ORF/pTP 3′UTR |
3 | miR-125b-5p | −1158 | UCCCUGAGACCCUAACUUGUGA | ACGGGUUAGGCUCUUGGGAGCU | 4990 | 5012 | pTP+Pol 3′UTR |
4 | miR-1307-5p | −1755 | UCGACCGGACCUCGACCGGCU | ACUCGGCGUGGCGUCGGUCGUG | 9680 | 9702 | pTP ORF |
5 | miR-151a-3p | −526 | CUAGACUGAAGCUCCUUGAGG | 10,154 | 10,176 | pTP ORF | |
miR-151a-5p | −738 | UCGAGGAGCUCACAGUCUAGU | 6151 | 6173 | Pol ORF/pTP 3′UTR | ||
miR-151a-5p | −610 | 6843 | 6865 | Pol ORF/pTP 3′UTR | |||
6 | miR-22-3p | −581 | AGUUCUUCAGUGGCAAGCUUUA | AAGCUGCCAGUUGAAGAACUGU | 4978 | 5000 | pTP+Pol 3′UTR |
7 | miR-27b-3p | −465 | AGAGCUUAGCUGAUUGGUGAAC | UUCACAGUGGCUAAGUUCUGC | 9721 | 9743 | pTP ORF |
8 | miR-29a-3p | −1138 | ACUGAUUUCUUUUGGUGUUCAG | UAGCACCAUCUGAAAUCGGUUA | 4234 | 4256 | pTP+Pol 3′UTR |
miR-29a-3p | −583 | 9850 | 9872 | pTP ORF | |||
9 | miR-423-3p | −707 | AGCUCGGUCUGAGGCCCCUCAGU | 5182 | 5204 | Pol ORF/3′UTR+ pTP 3′UTR | |
miR-423-3p | −702 | 7965 | 7987 | Pol ORF/pTP 3′UTR | |||
miR-423-3p | −698 | 5733 | 5755 | Pol ORF/pTP 3′UTR | |||
miR-423-3p | −713 | 10,265 | 10,287 | pTP ORF | |||
miR-423-5p | −737 | UGAGGGGCAGAGAGCGAGACUUU | 6751 | 6773 | Pol ORF/pTP 3′UTR | ||
miR-423-5p | −657 | 5528 | 5550 | Pol ORF/pTP 3′UTR | |||
miR-423-5p | −656 | 10,374 | 10,396 | pTP ORF | |||
10 | miR-7-5p | −1113 | UGGAAGACUAGUGAUUUUGUUGUU | CAACAAAUCACAGUCUGCCAUA | 6942 | 6964 | Pol ORF/pTP 3′UTR |
miR-7-5p | −559 | 6888 | 6910 | Pol ORF/pTP 3′UTR | |||
miR-7-5p | −1011 | 9545 | 9567 | pTP ORF | |||
11 | miR-99b-5p | −694 | CACCCGUAGAACCGACCUUGCG | CAAGCUCGUGUCUGUGGGUCCG | 6258 | 6280 | Pol ORF/pTP 3′UTR |
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Ausserhofer, P.; Kiss, I.; Witte, A.; Klein, R. Anti-Adenoviral Effect of Human Argonaute 2 Alone and in Combination with Artificial microRNAs. Cells 2024, 13, 1117. https://doi.org/10.3390/cells13131117
Ausserhofer P, Kiss I, Witte A, Klein R. Anti-Adenoviral Effect of Human Argonaute 2 Alone and in Combination with Artificial microRNAs. Cells. 2024; 13(13):1117. https://doi.org/10.3390/cells13131117
Chicago/Turabian StyleAusserhofer, Philipp, Izabella Kiss, Angela Witte, and Reinhard Klein. 2024. "Anti-Adenoviral Effect of Human Argonaute 2 Alone and in Combination with Artificial microRNAs" Cells 13, no. 13: 1117. https://doi.org/10.3390/cells13131117