Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.

Miguel Mata,Steven Neben,Jan Carette,Karim Majzoub,Paul A Khavari,Muthukumar Ramanathan,Peter Sarnow

doi:10.1371/journal.ppat.1007467

Abstract

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5’ end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5’ genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5’ end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5’ end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

Highlights

IntroductionMany cell- and virus-encoded microRNAs (miRNAs) regulate the expression of mRNAs by binding to the 3’ noncoding regions of target mRNAs. The binding is facilitated by an RNAinduced silencing complex (RISC) that mediates base-pair interactions between nucleotides two through seven in the microRNA (seed sequences) and their complementary sites in the target mRNA (seed-match sequences)
Many cell- and virus-encoded microRNAs regulate the expression of mRNAs by binding to the 3’ noncoding regions of target mRNAs
C3U hepatitis C virus (HCV) RNA replicates with higher efficiency than wild-type HCV RNA during miR-122 sequestration

Summary

Introduction

Many cell- and virus-encoded microRNAs (miRNAs) regulate the expression of mRNAs by binding to the 3’ noncoding regions of target mRNAs. The binding is facilitated by an RNAinduced silencing complex (RISC) that mediates base-pair interactions between nucleotides two through seven in the microRNA (seed sequences) and their complementary sites in the target mRNA (seed-match sequences). In the HCV genome, we discovered two binding sites for miR-122 at the 5’ proximal end of the viral RNA [3] Occupancy of both sites by miR-122 is required for the maintenance of viral RNA abundance in infected liver cells [3, 6,7,8]. Further evidence suggests that miR-122 participates in the switch of viral RNAs from the translation to the replication phase in the viral life cycle by displacing of RNA binding proteins that enhance viral mRNA translation [17]

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