Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

Miaoge Xue,Mark E Peeples,Yuanmei Ma,Stefan Niewiesk,Boxuan Simen Zhao,Zijie Zhang,Chuan He,Jianrong Li,Xueya Liang,Anzhong Li,Mijia Lu,Jiyong Zhou,Zhike Lu,Olivia Harder,Phylip Chen,Yunsheng Xu

doi:10.1038/s41467-019-12504-y

Abstract

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

Highlights

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes
To investigate whether respiratory syncytial virus (RSV) RNA contains m6A, RNA was extracted from highly purified virions grown in HeLa cells, and the purity of RNA was examined by real-time RT-PCR to ensure that they were free from any contamination of host RNAs and viral mRNAs (Supplementary Fig. 1)
We found that ~0.7% of the A bases were m6A methylated in RSV viral RNAs, a somewhat higher level than the host mRNAs (0.1–0.4%)

Summary

Introduction

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Studies from the early 1970s showed that viral RNAs of several DNA viruses, retroviruses, and influenza virus contained internal m6A modifications[4,24,25,26,27,28,29]. It is still controversial whether viral m6A positively or negatively regulates HIV replication[30,31,32], it was shown that m6A promotes gene expression of influenza virus[33] and simian virus 4034. The roles of m6A in viral virulence, pathogenesis, and immunity are not clear

Methods

Results

Conclusion