Impact of a Patient-Derived Hepatitis C Viral RNA Genome with a Mutated MicroRNA Binding Site

Abstract

25Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA 26 amplification in liver cells. This microRNA interacts with two different conserved sites at the very 27 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. 28Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound 29 loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a 30 viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide 31 from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of 32 replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, 33 when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in 34 the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, 35C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 36 abundance is high in the liver. These findings suggest that sequestration of miR-122 leads to a 37 resistance-associated mutation that has only been observed in treated patients so far, and 38 raises the question about the function of the C3U variant in the peripheral blood. 40Author Summary 41With the advent of potent direct-acting antivirals (DAA), hepatitis C virus (HCV) can now be 42 eliminated from the majority of patients, using multidrug therapy with DAAs. However, such 43 DAAs are not available for the treatment of most RNA virus infections. The main problem is the44 high error rate by which RNA-dependent RNA polymerases copy viral RNA genomes, allowing 45 the selection of mutations that are resistant to DAAs. Thus, targeting host-encoded functions 46 that are essential for growth of the virus but not for the host cell offer promising, novel 47 approaches. HCV needs host-encoded microRNA miR-122 for its viral RNA replication in the 48 liver, and depletion of miR-122 in HCV patients results in loss of viral RNA. This study shows 49 3 that a single-nucleotide mutation in HCV allows viral RNA amplification when miR-122 50 abundances are low, concomitant with changes in its tropism. 51 52 Introduction 53 54 Many cell-and virus-encoded microRNAs (miRNAs) regulate the expression of mRNAs by 55 binding to the 3' noncoding regions of target mRNAs. The binding is facilitated by an RNA-56 induced silencing complex (RISC) that mediates base-pair interactions between nucleotides two 57 through seven in the microRNA (seed sequences) and their complementary sites in the target 58 mRNA (seed-match sequences). This targeting event inhibits the translation of the mRNA. In 59 addition, deadenylation at the 3' of the mRNA, followed by decapping and 5' to 3' degradation of 60 the mRNA greatly increases its turnover [1, 2]. 61The growth of hepatitis C virus (HCV), a member of the flaviviridae, is dependent on the 62 most abundant miRNA in the liver, miR-122 [3]. In the liver...