Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo

Abstract

Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. 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. Despite major efforts, 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 the zinc binding activity of M2-1 is essential for virus replication and pathogenesis in vivo. Recombinant hMPVs that lacked zinc binding activity were not only defective in replication in the upper and lower respiratory tract but also triggered a strong protective immunity in cotton rats. Thus, inhibition of M2-1 zinc binding activity can lead to the development of novel, live attenuated vaccines, as well as antiviral drugs for pneumoviruses. Human metapneumovirus (hMPV), first characterized in 2001 in the Netherlands (1), belongs to the Metapneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae family. Since its discovery, hMPV has been isolated from individuals of all ages with acute respiratory tract infection, especially from infants, childr...

Aberrant T cell immunity triggered by human Respiratory Syncytial Virus and human Metapneumovirus infection

“…159 In addition, the M2-1 protein is critical for hMPV replication and pathogenesis through its Zinc binding activity, since a recombinant hMPV carrying mutations in the zinc binging motif was highly attenuated in cotton rats. 160 This is consistent with studies that demonstrate that hMPV lacking the M2-1 gene could not replicate in hamsters. 161 However, is not essential for hMPV infectivity and growth in vitro as deletion of the ORF of the M2-1 protein is dispensable for viral replication in VERO cells.…”

Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis

“…In contrast, deletion of M2-1 from the hRSV genome was lethal to the virus (6,18,19). Similar to hRSV M2-1, hMPV M2-1 incorporates zinc ions at a molecular ratio of 1:1 (21,22). However, the role of individual amino acids in the zinc binding motif (CCCH) differs between hMPV and hRSV M2-1.…”

“…The hMPV M2-1 gene was PCR amplified from a cDNA clone of strain NL/1/00 and was inserted into an E. coli expression system pGEX-4T-1 vector at BamHI and NotI sites (21). To facilitate protein purification, a glutathione S-transferase (GST) fusion protein was fused to the N terminus of the M2-1 gene.…”

“…In contrast, in hRSV M2-1, all four residues in the CCCH motif were equally important for zinc binding activity (H. Cai and J. Li, unpublished data). Finally, mutations to the zinc binding motif (CCCH) in hMPV M2-1 yielded viable recombinant viruses, demonstrating that zinc binding activity was not required for virus viability (21). In contrast, mutations to the zinc binding motif (CCCH) in hRSV M2-1 were lethal to the virus (19), suggesting that zinc binding is absolutely essential for the hRSV life cycle.…”

“…However, the role of individual amino acids in the zinc binding motif (CCCH) differs between hMPV and hRSV M2-1. In hMPV M2-1, the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) were found to be essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding (21). In contrast, in hRSV M2-1, all four residues in the CCCH motif were equally important for zinc binding activity (H. Cai and J. Li, unpublished data).…”