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Phosphorylation of enteroviral 2A^pro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

ABSTRACT: Enteroviral 2A proteinase (2A^pro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2A^pro ; however, the mechanism underlying the regulation of 2A^pro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2A^pro or Thr125 of coxsackievirus B3 2A^pro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2A^pro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2A^pro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2A^pro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2A^pro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2A^pro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

SUBMITTER: Wang Y

PROVIDER: S-EPMC10107306 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Phosphorylation of enteroviral 2A<sup>pro</sup> at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Wang Yuya Y Zou Wenjia W Niu Yan Y Wang Sanyuan S Chen Bangtao B Xiong Rui R Zhang Peng P Luo Zhijun Z Wu Yong Y Fan Changfa C Zhong Zhaohua Z Xu Ping P Peng Yihong Y

Journal of medical virology 20230101 1

Enteroviral 2A proteinase (2A<sup>pro</sup> ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential ...[more]

PMID: 36511115

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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.

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Phosphorylation of enteroviral 2A^pro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Publications

Phosphorylation of enteroviral 2A<sup>pro</sup> at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

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Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Publications

Phosphorylation of enteroviral 2A<sup>pro</sup> at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

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OmicsDI is part of the ELIXIR infrastructure

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Phosphorylation of enteroviral 2A^pro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.