Le rôle de la région variable du 3’ UTR dans la réplication du virus de l’hépatite C
The hepatitis C virus (HCV), a member of the Flaviviridae, contains a single (+) strand linear RNA genome which incorporates one long open reading frame flanked by two untranslated regions known as the 5’UTR and 3’UTR. These regions contain structures known to function in the regulation of HCV translation and replication. The 3’UTR is the focus of this research project. Its tripartite structure consists of a variable region (VR), a poly (U/UC) region and the X-tail. Our laboratory recently identified a VR sequence forming a duplex structure with a distal sequence located in the NS5B gene. Because NS5B encodes the viral polymerase, and seeing that the 3’UTR is involved in viral replication, we postulated that this duplex may function in controlling the level of HCV RNA replication. To test this hypothesis, we constructed HCV 1b mutants in the respective VR and NS5B annealing sequences to disrupt complementarity and prevent duplex formation. We also created a mutant with complementary mutations at both sites to reconstitute the duplex. RNA from these constructs was synthesized in vitro and transfected into Huh7.5 cells. Following a 9-day incubation period, RNA from all transfected cultures was harvested and HCV RNA was quantified by RT-qPCR. The data indicate an increase of 5.3X and 3.4X for HCV viral RNA harvested from cultures with disrupted 3’UTR-VR and NS5B, respectively, relative to wild-type HCV 1b, whereas the reconstituted HCV construct yielded a 2.6X increase in viral RNA. While these figures do not reach statistical significance, they suggest that the duplex structure modulates HCV replication downward. Further pursuit of this work is necessary to better elucidate the role of the VR-NS5B duplex structure on HCV replication.
Flaviviridae 3’UTR VR NS5B Interactions distales HCV Distal interactions Replication RNA structures