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. IMPORTANCEThe 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. Human metapneumovirus (hMPV) is one of the leading causes of upper and lower respiratory tract infection in humans, particularly in infants, young children, the elderly, and immunocompromised individuals. The virus was first identified in children suffering from respiratory illness in the Netherlands in 2001 (1). Subsequent epidemiological studies suggested that 5 to 15% of all respiratory tract infections in infants and young children were caused by hMPV, a proportion second only to that of human respiratory syncytial virus (hRSV) (2-5). hMPV belongs to the Metapneumovirus genus within the Pneu...

A conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence

“…The virion host shutoff (vhs) protein is the key regulator of the induced early host shutoff response, and herpes simplex viruses with vhs deleted have been proposed as live attenuated vaccines (25,26). Human metapneumovirus protein M2-1 phosphorylation plays important regulatory roles in RNA synthesis, replication, and pathogenesis, and inhibition of M2-1 phosphorylation may serve as a novel approach for development of live attenuated vaccines (27).…”

Human Metapneumovirus: Mechanisms and Molecular Targets Used by the Virus to Avoid the Immune System

“…Little is known about the role of the M2-1 protein in hMPV evasion mechanisms. However, this protein presents a critical role in hMPV pathogenesis and viral replication ( 47 , 48 ). One possible characteristic suggested for the protein's role is its Zinc binding activity.…”

“…Once TLR3 detects hMPV, it is able to activate IFN regulatory factor 3 (IRF3) through TRIF, whereas TLR7 activates IRF7 through MyD88 ( 41 , 52 ). RIG-I and MDA5 are two helicases from the RIG-I-like receptor (RLR) family that induce signaling downstream to MAVS after detecting viral dsRNA in the cytosol ( 48 , 56 , 57 ). Subsequently, MAVS activates IRF3, and this in turn activates the production of type-I IFN and the up-regulation of IRF7 and NF-κB, thus producing proinflammatory cytokines and type-III IFNs ( 80 ).…”

Interferon-Mediated Response to Human Metapneumovirus Infection

“…The phosphoprotein (P), nucleocapsid (N), and viral polymerase (L) are essential proteins for viral RNA synthesis [ 6 , 10 , 11 ]. M2-1 protein appears to be necessary for HMPV replication in vivo [ 6 , 12 ]. HMPV has been classified into two main groups, i.e., A and B, and further subdivided into A1, A2, B1, and B2, based on genetic analysis [ 8 , 13 , 14 , 15 ].…”