Understanding the mechanism and annealing requirements for miR-122 and other small RNA promotion of Hepatitis C Virus
Date
2021-03-15
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-9933-9981
Type
Thesis
Degree Level
Doctoral
Abstract
Hepatitis C Virus (HCV) is a flavivirus that causes chronic infections of the liver and can lead to liver cirrhosis and hepatocellular carcinoma. HCV has a 9.6 kb long positive-sense RNA genome that consists of the 5’ untranslated region (UTR), a polyprotein coding region, and a 3’UTR region. The polyprotein encodes both structural proteins that form the virion and nonstructural proteins that are required for virus replication and the UTRs regulate genome translation and replication. Replication of the viral genome also requires annealing of a liver-specific microRNA, miR-122 to 2 sites within the 5’ untranslated region (UTR) but the mechanism by which it promotes HCV lifecycle is unclear. We therefore sought to determine the role of miR-122 in the HCV lifecycle. In this study we show that location specific annealing of small RNAs to the miR-122-binding region promotes translation, stabilizes the genome and supports HCV replication as miR-122 mimics, and we speculate that annealing of miR-122 and the small RNAs stimulates formation of the Internal Ribosomal Entry Site (IRES). We further show that miR-122 colocalizes with HCV at both early and later time points suggesting a role of miR-122 throughout HCV infection. We first aimed to analyze the importance of miR-122 binding site sequences, by mutagenizing the HCV 5’UTR proximal conserved region and analyzing how point mutations affect viral genomic RNA replication capabilities. By using siRNAs to pressure escape mutations, replication-competent HCV genomes having nucleotide changes in the conserved 5’ untranslated region (UTR) were identified. Interestingly, some mutant viruses had also adapted to use the siRNA as a miR-122- mimic. Further, when siRNA cleavage activity was blocked by using Ago2 knockout cells, all of the siRNAs used to pressure evolution of escape mutants promoted wild type virus replication, equally efficiently as miR-122, and could stabilize the viral genome. We therefore showed that a.) annealing of small interfering RNAs (siRNA) to the HCV 5’ UTR can mimic the pro-viral activity of miR-122, b.) replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication c.) HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5’ end annealing or 3’ overhanging nucleotides and that d.) that the complex annealing pattern formed by miR-122 on the HCV genome was not required for the pro-viral mechanism. Our findings also provided a method for us to assess the impact on HCV replication by small RNA annealing to other locations on the genome. Using a panel of HCV genome-targeting siRNAs, we found that annealing between nucleotides 1 and 44 in HCV 5’UTR, promoted HCV replication, and annealing within the IRES, NS5B and 3’UTR regions did not. An siRNA that targeted nucleotides 19-37 promoted replication most efficiently and more effectively than miR-122 and we defined the minimum nucleotide annealing required to promote HCV as nucleotides 23-35. Replication efficiency decreased as the siRNA target site moved away from this region. We also found that the difference in replication efficiencies induced by the panel of siRNAs correlated with their ability to stimulate translation and their predicted ability to induce the canonical SLII structure within the HCV IRES. Finally, like miR-122, annealing of the siRNAs to the 5’ UTR also stabilized the viral genome, but the siRNAs did so regardless of whether they could promote replication or not, suggesting that genome stabilization alone is insufficient for HCV replication. To gain more insight into when miR-122 functions during the HCV lifecycle, we monitored the interaction of miR-122 with the HCV genome by Fluorescence in-situ hybridization (FISH). Our data suggests that HCV colocalizes with miR-122 at early time points after transfection, supporting the concept of an involvement in early stages of the virus infection cycle. However, colocalization was also evident at later stages and suggest possible roles in ongoing virus replication, virus assembly and/or egress. Thus, we have defined the RNA domain and the double stranded RNA element formed by small RNA annealing that is pivotal for enhancement of virus amplification. We were also able to observe HCV and miR-122 colocalization at various time points suggesting potential roles throughout all stages of the viral replication cycle. Thus, we present a model that miR-122 annealing, promotes HCV replication primarily by activating the HCV IRES and stimulating translation. We also suggest that genome stabilization by miR-122 is important and contributes to virus replication, it is not sufficient alone to activate HCV replication by miR-122 Further given its critical role in promoting translation, we propose that miR-122 interacts with the HCV genome while it is in association with the endoplasmic reticulum and is required early in the lifecycle but also a later times to maintain an HCV infection.
Description
Keywords
Hepatitis C Virus, microRNA-122, Argonaute, siRNAs, translation
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology