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

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.

SUBMITTER: Xue M

PROVIDER: S-EPMC6785563 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Xue Miaoge M Zhao Boxuan Simen BS Zhang Zijie Z Lu Mijia M Harder Olivia O Chen Phylip P Lu Zhike Z Li Anzhong A Ma Yuanmei Y Xu Yunsheng Y Liang Xueya X Zhou Jiyong J Niewiesk Stefan S Peeples Mark E ME He Chuan C Li Jianrong J

Nature communications 20191009 1

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 transcri ...[more]

PMID: 31597913

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Project description:UnlabelledHuman metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivoImportanceThe pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus replication and pathogenesis in vivo Recombinant hMPVs lacking phosphorylation in M2-1 exhibited limited replication in the upper and lower respiratory tract and triggered strong protective immunity in cotton rats. This work highlights the important role of M2-1 phosphorylation in viral replication and that inhibition of M2-1 phosphorylation may serve as a novel approach to develop live attenuated vaccines as well as antiviral drugs for pneumoviruses.

Dataset Information

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

Publications

Viral N<sup>6</sup>-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus.

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