title: Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.
abstract: Human 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 vivo
  
  
  
  The 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.
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Cai
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Laboratory of New Drugs Safety Evaluation, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, People's Republic of China.
authors.forename: Mijia
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Xueya
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Jennings
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Niewiesk
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA li.926@osu.edu.
authors.forename: Jianrong
authors.initials: J
authors.lastname: Li
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D029121:Metapneumovirus / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D018184:Paramyxoviridae Infections / Q000473:pathology / Q000821:virology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D059386:Reverse Genetics; D016205:Sigmodontinae; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D014774:Virulence; D014779:Virus Replication
chemical_list: D050505:Mutant Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014763:Viral Matrix Proteins