M基因影响流感病毒对哺乳动物致病性的分子机制

Influenza is an important zoonotic disease for humans and animals. The viral pathogenesis of influenza virus is associated with multiple genes and their encoded proteins. The M gene influence on pathogenesis is crucial, but the mechanisms of how M gene affects pathogenesis are still not completely understood. Our previous studies demonstrate that the chimeric bat influenza virus containing M gene from either the human origin PR8/H1N1 virus or swine origin TX98/H3N2 virus, which is highly identical to each other, showed significantly difference in pathogenicity in mice; which provides a solid foundation for us to elucidate the mechanisms of M gene influencing viral pathogenicity. In this proposal, we will investigate whether the M gene of the H9N2 virus, which has contributed internal genes to important zoonotic influenza viruses such as H5N1 and H7N9 viruses, also enhances virulence in mice in the genetic backbone of the chimeric bat influenza virus. Meanwhile, we will identify the key regions and amino acids in the M gene-encoded proteins critical for viral pathogenesis by using sequence comparison, partial replacement, multiple and single mutation, virus rescue and animal pathogenicity studies. Molecular mechanisms of M gene influencing viral pathogenesis will be elucidated through studying interaction of M1/M2 proteins with viral proteins（HA and NA）, the formation efficiency of virus like particles (VLPs) using different M1/M2, VLP morphology under the electron microscope, the co-localization of M1/M2 with HA and NA by the confocal microscope, how different M1/M2 influences viral replication (vRNA) and transcription (mRNA) level and viral packaging efficiency. Our results will provide novel insights on biology of influenza virus and understanding of molecular mechanisms of M gene influencing viral pathogenicity, resulting in necessary scientific knowledge for prevention and control of influenza.

流感是重要人畜共患病，严重威胁人类和动物健康。流感病毒的致病性是由多个基因及其编码蛋白决定的，M基因及其编码蛋白发挥着重要作用，但其分子机制尚不完全清楚。我们前期研究发现蝙蝠流感病毒M基因被同源性很高的两个哺乳动物来源M基因（人PR8/H1N1和猪TX98/H3N2）替换后的重组病毒在小鼠体内展现出显著的致病性差异。前期研究结果为我们进一步阐明M基因影响流感病毒致病性的分子机制奠定了坚实的基础。本项目将继续研究禽源H9N2病毒的M基因是否影响流感病毒对哺乳动物的致病性，结合前期研究发现的差异氨基酸位点，通过序列比对、部分替换、定点突变、病毒拯救和动物试验等方法确定M基因影响流感病毒致病性的关键区域和关键氨基酸位点，研究其如何影响与病毒蛋白间的相互作用、病毒样颗粒形成及形态、病毒核酸复制转录和包装效率，进而来阐明M基因影响流感病毒对哺乳动物致病性的分子机制，为流感病毒防控提供新的科学依据。

流感是重要的人兽共患病，严重威胁人类和动物的健康。流感病毒的致病性是由多个基因及其编码蛋白决定的，M基因及其编码的蛋白发挥着重要作用。M基因参与致病性影响的分子机制仍不清楚。本项目在前期研究基础上，以嵌合蝙蝠流感病毒为背景，对禽源、蝙蝠源病毒M基因的影响、以及M基因影响病毒体内外复制和小鼠致病性的关键分子基础进行了研究。研究结果表明：禽源M基因的重组病毒在哺乳动物宿主细胞和小鼠体内复制较差，蝙蝠源M基因的重组病毒在哺乳动物宿主细胞上复制比禽宿主细胞上显著提高。具有一致禽源M基因与禽源表面基因的重组病毒在禽宿主细胞上的复制比相同背景的蝙蝠源M基因的重组病毒要高，进一步地研究表明这可能与前者增加了对禽源受体（α 2,3-SLN）的结合能力有关。M1蛋白的差异对于病毒在细胞上复制的影响要明显于M2蛋白的差异，其中M1-41位， M2-27位氨基酸的差异对于重组流感病毒在细胞复制和小鼠体内的致病性有重要决定作用。本项目研究结果提示，不同来源M基因对于流感病毒的复制和宿主嗜性有影响作用，这可能是流感病毒分别在哺乳动物宿主和禽类宿主体内长期进化适应的结果；另外，我们也解析了影响病毒复制和小鼠致病性的M蛋白关键分子基础(M-41, M2-27），为流感病毒的研究与防控提供了重要知识信息。