Cai Hui, Zhang Yu, Ma Yuanmei, Sun Jing, Liang Xueya, Li Jianrong
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA
J Virol. 2015 Jun;89(12):6391-405. doi: 10.1128/JVI.03488-14. Epub 2015 Apr 8.
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.
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. 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.
人偏肺病毒（hMPV）是副粘病毒科肺病毒亚科的成员，可引起人类呼吸道感染。与肺病毒亚科的成员不同，肺病毒的聚合酶复合物需要一种额外的辅助因子，即M2-1蛋白，它作为转录抗终止因子发挥作用。已发现M2-1蛋白结合锌离子，尽管锌结合活性在病毒复制和发病机制中的具体作用尚不清楚。在本研究中，我们发现hMPV M2-1锌结合基序（CCCH）中的第三个半胱氨酸（C21）和最后一个组氨酸（H25）对锌结合活性至关重要，而前两个半胱氨酸（C7和C15）在锌结合中仅起次要或冗余作用。此外，锌结合基序对M2-1的寡聚化至关重要。随后，回收了在锌结合基序中携带突变的重组hMPV（rhMPV）。有趣的是，缺乏锌结合活性的rhMPV-C21S和-H25L突变体在细胞培养中的复制延迟，并且在棉鼠中高度减毒。相比之下，保留60%锌结合活性的rhMPV-C7S和-C15S菌株在棉鼠中的复制效率与rhMPV一样高。重要的是，缺乏锌结合活性的rhMPV引发了高水平的中和抗体，并提供了针对rhMPV攻击的完全保护。综上所述，这些结果表明锌结合活性对于体内病毒复制和发病机制是不可或缺的。这些结果还表明，抑制锌结合活性可能是一种合理减毒hMPV以及可能其他肺病毒以用于疫苗目的的新方法。
肺病毒包括许多重要的人类和动物病原体，如人呼吸道合胞病毒（hRSV）、hMPV、牛呼吸道合胞病毒和禽偏肺病毒（aMPV）。在这些病毒中，hRSV和hMPV是婴幼儿急性呼吸道感染的主要原因。尽管付出了巨大努力，但仍没有抗病毒药物或疫苗来对抗这些疾病。所有已知的肺病毒都编码一种锌结合蛋白M2-1，它是一种转录抗终止因子。在这项工作中，我们发现M2-1的锌结合活性对于体内病毒复制和发病机制至关重要。缺乏锌结合活性的重组hMPV不仅在上呼吸道和下呼吸道的复制有缺陷，而且在棉鼠中引发了强烈的保护性免疫。因此，抑制M2-1锌结合活性可导致开发新型减毒活疫苗以及用于肺病毒的抗病毒药物。
