Cai Hui, Zhang Yu, Lu Mijia, Liang Xueya, Jennings Ryan, Niewiesk Stefan, Li Jianrong
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA.
Laboratory of New Drugs Safety Evaluation, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang, People's Republic of China.
J Virol. 2016 Jul 27;90(16):7323-7338. doi: 10.1128/JVI.00755-16. Print 2016 Aug 15.
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.
人偏肺病毒（hMPV）是全球范围内婴儿、老年人和免疫功能低下个体上、下呼吸道感染的主要病原体。与所有肺病毒一样，hMPV编码锌结合蛋白M2-1，其在RNA合成中发挥重要调节作用。M2-1蛋白被磷酸化，但其磷酸化在病毒复制和发病机制中的具体作用仍不清楚。在本研究中，我们发现hMPV M2-1在氨基酸残基S57和S60处被磷酸化。随后的诱变发现，磷酸化对于锌结合活性和寡聚化并非必不可少，而锌结合活性的抑制则消除了M2-1蛋白的磷酸化和寡聚化。使用反向遗传学系统，回收了在M2-1蛋白中缺少一个或两个磷酸化位点的重组hMPV（rhMPV）。这些重组病毒在基因组RNA复制和mRNA转录方面均显著下降。此外，这些重组病毒在细胞培养和棉鼠中高度减毒。重要的是，M2-1蛋白缺乏磷酸化的rhMPV引发高水平的中和抗体，并提供针对野生型hMPV攻击的完全保护。总体而言，这些数据表明M2-1蛋白的磷酸化上调了hMPV在体内的RNA合成、复制和发病机制。
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。目前，尚无对抗这些疾病的抗病毒药物或疫苗。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的磷酸化对于病毒在体内的复制和发病机制至关重要。M2-1缺乏磷酸化的重组hMPV在上、下呼吸道中的复制有限，并在棉鼠中引发强烈的保护性免疫。这项工作突出了M2-1磷酸化在病毒复制中的重要作用，并且抑制M2-1磷酸化可能作为开发减毒活疫苗以及肺病毒抗病毒药物的一种新方法。
