N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

Nandan S Gokhale,Sharon E Hopcraft,Stacy M Horner,Christine Vazquez,Kendra M Quicke,Alexa B.R Mcintyre,Brittany A Law,Matthew J Evans,Olga R Ilkayeva,Shelton S Bradrick,Jason Willer,Christopher L Holley,Mariano A Garcia-Blanco,Mehul S Suthar,Edward M Kennedy,Jorge A Gandara,Allison E Roder,Christopher E Mason,Michael J Mcfadden

doi:10.1016/j.chom.2016.09.015

Abstract

SummaryThe RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Highlights

The chemical modification of RNA is an important post-transcriptional regulator of RNA
The m6A Machinery Regulates hepatitis C virus (HCV) Particle Production To determine whether m6A regulates HCV infection, we depleted the m6A methyltransferases METTL3 and METTL14 (METTL3+14) by small interfering RNA in Huh7 liver hepatoma cells and infected these cells with HCV
Immunoblot analysis of cell extracts harvested at 72 hr post-infection revealed that METTL3+14 depletion significantly increased the abundance of the HCV NS5A protein, a marker of viral replication, relative to its level in cells treated with non-targeting control small interfering RNA (siRNA) (Figure 1A)

Summary

Introduction

The chemical modification of RNA is an important post-transcriptional regulator of RNA. Flaviviridae, including Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and hepatitis C virus (HCV), represent both established and emerging pathogens. They contain a positivesense, single-stranded RNA genome that encodes a viral polyprotein and use similar replication strategies. RNA-based regulation of these viral genomes plays a fundamental role in their infection, such as the liver-specific miRNA miR-122 for HCV replication, RNA structural elements for HCV and DENV replication, and 20-O methylation of the 50 cap of WNV RNA for immune evasion and WNV replication (Bidet and GarciaBlanco, 2014; Hyde et al, 2014; Jopling et al, 2005; Mauger et al, 2015; Pirakitikulr et al, 2016)

Results

Discussion

Conclusion