Interference by Defective influenza Viruses

描述(申请人提供)：流感病毒(正粘病毒)包括一组主要的人类和动物病原体，属于被膜的、节段性的、负链的RNA病毒。甲型流感病毒的7号片段RNA编码两种重要的结构蛋白--来自非剪接mRNA的M1和来自剪接mRNA的M2。M1是一种相对较小、高度保守的蛋白(A型病毒为252个氨基酸，B型病毒为248个氨基酸)。M1是病毒颗粒中含量最丰富的蛋白质，在病毒复制的许多方面起着关键作用。这些包括(I)在病毒进入和剥离过程中M1从M1/vRNP复合体中解离，(Ii)M1的核进入，(Iii)M1与vRNP相互作用以形成M1/vRNP复合体，(Iv)M1在vRNP从细胞核退出到细胞质中的作用，(V)M1与病毒糖蛋白(HA、NA和M2)的相互作用，(Vi)M1的膜结合，(Vii)M1的二聚体/低聚物的形成，(Viii)M1在病毒萌发中的作用，包括(A)病毒成分在组装部位的招募，(B)为病毒颗粒的萌发和释放招募宿主成分；(C)病毒形态。尽管关于M1实现这些功能的具体机制存在相互矛盾的假设和结果，但M1必须拥有特定的基序或结构域才能实现这些功能。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：i.核定位基序，ii。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/齐聚基序，vi.其他与晚期(L)相似的结构域包括宿主蛋白招募基序。我们的初步数据表明，在流感病毒M1中存在一个L结构域样基序。八.此外，我们还计划研究M1/vRNP复合体或vRNP单独在选择顶膜出芽位置方面是否起作用，因为我们和其他人已经证明，主要糖蛋白HA不是流感病毒顶芽的主要决定因素。我们计划使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为开发活病毒疫苗而产生稳定的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.