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

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.

Host-derived circular RNAs display proviral activities in Hepatitis C virus-infected cells

“…Plasmid HCV J6/JFH-1 Rluc genotype 2 contains a renilla firefly reporter, and plasmid HCV H77S.3/GLuc genotype 1a carries a gaussia luciferase reporter [74,75]. Plasmids were linearized [74] and transcribed using the MEGAscript T7 Transcription Kit (ThermoFisher) according to the manufacturer's protocol. Huh7.5 Sec14L2 cells were transfected with siRNA duplexes at 50 nM final concentration at day 1.…”

“…The following day, the cells were transfected with 1 μg of in vitro-transcribed J6/JFH-1 Rluc RNA or H77S.3/GLuc RNA using the TransIT mRNA transfection kit (Mirus Bio LLC) according to the manufacturer's protocol. Renilla and gaussia luciferase activities were measured as described [74], using a Glomax 20/20 luminometer for 10 second integration time.…”

Host-derived Circular RNAs Display Proviral Activities in Hepatitis C Virus - Infected Cells

“…Plasmid HCV J6/JFH-1 Rluc genotype 2 contains a renilla firefly reporter, and plasmid HCV H77S.3/GLuc genotype 1a carries a gaussia luciferase reporter [75, 76]. Plasmids were linearized [75] and transcribed using the MEGAscript T7 Transcription Kit (ThermoFisher) according to the manufacturer’s protocol. Huh7.5 Sec14L2 cells were transfected with siRNA duplexes at 50 nM final concentration at day 1.…”

“…The following day, the cells were transfected with 1 μg of in vitro-transcribed J6/JFH-1 Rluc RNA or H77S.3/GLuc RNA using the TransIT mRNA transfection kit (Mirus Bio LLC) according to the manufacturer’s protocol. Renilla and gaussia luciferase activities were measured as described [75], using a Glomax 20/20 luminometer for 10 second integration time.…”

The Role of the Liver-Specific microRNA, miRNA-122 in the HCV Replication Cycle

“…Sequence analysis of viral RNAs obtained from serum of several of the patients treated with miR-122 antagonists suggest the emergence of resistance. Sequencing revealed a resistance-associated substitution of a uridine for a cytidine nucleotide 3 (C3U) [ 101 ]. This mutation occurred in the auxiliary region of miR-122 binding site 1 and biochemical assays showed reduced binding of miR-122 at site 1 and destabilization of the tri-molecular structure formed by 2 copies of miR-122 and the HCV RNA.…”