RNA interference : impact on RNA virus replication and use to study virus-host interaction

MicroRNAs are short double-stranded RNAs of about 21 base pairs, which inhibit protein synthesis by binding target mRNA displaying sequence complementarity. microRNA can also hybridize to mRNA or genomic RNA of viruses in order to restrict their replication. It is well documented that microRNAs are involved in DNA virus biology. Many DNA viruses produce microRNA that regulate either viral or host gene expression. Certain DNA viruses also harness host microRNAs to regulate their replication or to modulate cellular immune responses. However, the role of cellular microRNAs in RNA virus biology is still debated. It is proposed that microRNAs contribute to cellular innate immunity against RNA viruses. In the first part of my thesis, we assessed the general influence of cellular microRNAs on Theiler’s virus replication. To do so, we developed a system to inactivate the Dicer gene, which encodes a crucial enzyme that participates to microRNA synthesis. Then, we compared virus replication in Dicer-positive and Dicer-negative cells. Transient Dicer inactivation led to a significant decrease of microRNA levels and to a strong increase of Vesicular stomatitis virus replication. In contrast, microRNAs depletion did not increase Theiler’s virus replication. In the second part of my thesis, we evaluated the effect of a miR-142-3p target sequence present in human rotavirus genome. Our results indicate that this microRNA does not inhibit, nor enhance the replication of an attenuated human rotavirus, although this microRNA binds the target sequence located in the viral genome. Lastly, we used the RNAi technology to analyse the antiviral activity of a murine interferon-inducible GTPase called Gvin1. This protein has a molecular mass of 280 kDa and is the largest known GTPase in any species. The GTP-binding domain of Gvin1 shares sequence similarity with those of other interferon-inducible GTPases, such as Mx and guanylate-binding proteins, which exhibit an antiviral activity against certain viruses. To study the putative antiviral function of Gvin1, we inhibited its expression by using a short hairpin RNA expressed from a lentiviral construct. Next, we compared the replication of RNA and DNA viruses in cells where Gvin1 expression was restricted or not. We showed that the inhibition of Gvin1 expression in interferon-treated cells increases the infection of Theiler’s virus, Vesicular stomatitis virus and, in particular, Murid herpesvirus-4. Moreover, Gvin1 impacts the nucleic acids uptake mediated by cellular transfection and rearranges the actin cytoskeleton. These results, however, need to be confirmed with other constructs, to rule out that they resulted from off-target effects. All together, our results suggest that Gvin1 plays a role in cellular resistance to viral infection by interfering with virus entry into cells.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201