miR-122-independent propagation of Hepatitis C Virus: Understanding the underlying mechanism through characterization of viral replication.
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Hepatitis C Virus (HCV) is a serious global health problem, infecting almost 3% of the world’s population. Infection with the Hepatitis C virus (HCV) leads to chronic liver infections in an estimated 70% of infected individuals and can lead to the development of various cancers, liver cirrhosis, and other complications late in chronicity. A liver-specific microRNA, miR-122, that binds to the 5’ UTR of the HCV genome plays an important role in the HCV life cycle by positively modulating the virus lifecycle. Work from our lab demonstrates that, unlike the wild-type HCV genome, some full-length RNAs with mutations in the 5’ UTR and bi-cistronic HCV replicons containing an additional IRES can replicate at low rates in miR-122-deficient cells. Although there are reports of miR-122-independent replication of HCV, no mechanism for independent replication has been proposed. In this study, we hypothesize that an alteration in translation regulation affects viral propagation and, in turn, promotes miR-122-independent replication. We observed that the genomes in which translation is regulated by the EMCV IRES and genomes having mutations in the 5’UTR that enhance HCV genome translation efficiency also promoted miR-122-independent replication. These findings support a role for miR-122 in regulating HCV translation. In silico structural analysis of miR-122 bound 5’UTR of HCV shows that it forms the stem-loops required for HCV IRES activity, while the unbound RNA was predicted to form an alternative RNA structure. Since RNA structures are dynamic, we speculate that miR-122 binding to the viral 5’UTR stabilizes the active IRES structure making it thermodynamically stable. The mutants that can replicate independently of miR-122 are also predicted to form the canonical structures even in the absence of miR-122. Thus, we suggest that the mutations on the 5’UTR might be shifting the equilibrium towards thermodynamically stable active IRES structure, and conferring enhanced translation efficiency by altering IRES activity or by providing better stability and enabling the genome to propagate inside the cell in the absence of any microRNA. Analysis of cells supporting miR-122-independent and dependent HCV replication by microscopy and flow cytometry revealed that cells supporting miR-122-independent replication expressed HCV proteins at levels similar to that seen during miR-122-dependent replication, but that the numbers of cells supporting HCV are smaller. These results suggest that the establishment of replication in a high proportion of cells requires miR-122, but for genomes capable of miR-122-independent HCV replication, the infection was only established in a few cells that might have received stochastically more RNA at the beginning of the infection. Further, we saw no evidence of virus adaptation to growth without miR-122, and in time course studies we also show that miR-122 supplementation or antagonization has a smaller influence on HCV replication after an infection has been established but had no effect on ongoing miR-122 independent replication of the virus. Hence, we suggest miR-122 plays an important role at the initial stage of infection by promoting viral translation, but it appears to have a smaller influence on the maintenance of infection of the virus. Altogether, we suggest that miR-22 induced enhanced translation or stability might be required at the initial stage of HCV infection to establish the infection, but can be dispensable for ongoing infection. However, when present, it assists the ongoing infection and might be doing so by helping the virus to establish new replication complexes in the dividing cells.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentMicrobiology and Immunology
ProgramMicrobiology and Immunology
SupervisorWilson, Joyce A
CommitteeChelico, Linda; van den Hurk, Sylvia; Kobryn, Kerri; Zhou, Yan
Copyright DateJanuary 2021
Hepatitis C Virus, miR-122, HCV mutants, miR-122-independent replications