Interference by Defective influenza Viruses

描述（由申请人提供）：流感病毒（正黏液病毒）包含一大类人类和动物病原体，属于包膜、分段、负链RNA病毒。甲型流感病毒的第7段RNA编码两个重要的结构蛋白——来自未剪接的mRNA的M1和来自剪接的mRNA的M2。M1是一种相对较小、高度保守的蛋白（a型病毒为252aa， B型病毒为248aa）。M1是病毒颗粒中含量最多的蛋白，在病毒复制的许多方面起着关键作用。这些包括(i)离解的M1 M1 / vRNP复杂的病毒,在入口和脱壳(ii)核条目M1,(3)交互与vRNP M1形成M1 / vRNP复杂,(iv) vRNP M1的角色退出核进入细胞质,(v)交互的M1病毒糖蛋白(HA、NA和M2), (vi)膜绑定的M1,(七)二聚体/低聚物形成的M1,(八)M1在病毒出芽病毒成分包括(a)招聘大会网站,(b)为出芽和释放病毒颗粒而招募宿主成分，(c)病毒形态。尽管关于M1完成这些功能的具体机制存在相互矛盾的假设和结果，但M1必须具有执行这些功能的特定基序或结构域。我们在这个项目中的具体目标是研究M1的以下基序在病毒生物学中的作用：1 .核定位基序；膜结合基序，iii。RNA/RNP结合基序，iv.锌指基序，v.糖蛋白（HA， NA, M2）结合基序，vi.二聚体/寡聚基序，vii。其他类似于晚(L)结构域的包括宿主蛋白募集基序。我们的初步数据表明在流感病毒M1中存在L结构域样基序。8。此外，我们还计划研究M1/vRNP复合物或vRNP本身是否在选择顶端膜的出芽位点中起任何作用，因为我们和其他人已经表明，HA（主要糖蛋白）不是流感病毒顶端出芽的主要决定因素。我们计划利用分子生物学和反向遗传学的强大工具来识别这些基序并确定它们在病毒生命周期中的功能。鉴定这些基序并阐明它们在病毒生物学中的功能，对于开发用于治疗的小干扰分子和产生用于开发活病毒疫苗的稳定减毒病毒突变体具有重要意义。

DESCRIPTION (provided by applicant): Influenza viruses (Orthomyxoviruses) encompass a major group of human and animal pathogens and belong to enveloped, segmented, negative stranded RNA viruses. The segment #7 RNA of influenza A viruses encodes two important structural proteins- M1 from unspliced mRNA and M2 from a spliced mRNA. M1 is a relatively small, highly conserved protein (252 aa in type A and 248 aa in type B viruses). M1 is the most abundant protein in virus particles and plays critical roles in many aspects of virus replication. These include (i) dissociation of M1 from the M1/vRNP complex during the entry and uncoating of virus, (ii) nuclear entry of M1, (iii) interaction of M1 with vRNP to form M1/vRNP complex, (iv) role of M1 in the exit of vRNP from nucleus into cytoplasm, (v) interaction of M1 with viral glycoproteins (HA, NA and M2), (vi) membrane binding of M1, (vii) dimer/oligomer formation of M1, (viii) role of M1 in virus budding including (a) recruitment of viral components at the assembly site, (b) recruitment of host components for budding and release of virus particles, (c) virus morphology. Although there are conflicting hypotheses and results on the specific mechanism by which M1 accomplishes many of these functions, M1 must possess specific motifs or domains for carrying out these functions. Our specific objectives in this project are to investigate the role of the following motifs of M1 in virus biology: i. Nuclear localization motif, ii. Membrane binding motifs, iii. RNA/RNP binding motif, iv. Zinc finger motif, v. Glycoprotein (HA, NA, M2) binding motifs, vi. Dimer/oligomerization motif, vii. Others similar to late (L) domain including host protein recruitment motifs. Our preliminary data suggest the presence of an L domain-like motif in influenza virus M1. viii. In addition, we also plan to investigate if M1/vRNP complex or vRNP alone plays any role in selecting the budding site at the apical membrane since we and others have shown that HA, the major glycoprotein, is not the major determinant in apical budding of influenza virus. We plan to use the powerful tools of molecular biology and reverse genetics in identifying these motifs and defining their functions in the virus life cycle. Identification of these motifs and elucidation of their functions in virus biology will be important towards developing small interfering molecules for use in therapy and generating stable attenuated virus mutants for the development of live virus vaccine.