Investigation of influenza virulence mediated by the NS1A protein

描述(由申请人提供)：流感病毒(正粘病毒科)在人类中引起一种高度传染性的呼吸道疾病，导致美国每年约36,000人死亡。这项研究的长期目标是通过体外、体内和小鼠动物模型揭示流感非结构蛋白-1(NS1A)蛋白影响病毒毒力的机制。推动这项研究的假设是，NS1A蛋白包含一个以前未确定的参与病毒毒力的位置。我们认为这个区域是一个核输出信号(NES)，抑制这个NES通过促进NS1A核功能来增强病毒的毒力。这一假说基于以下观察：1)表达NS1蛋白并在L77位进行单一丙氨酸替换的A型流感重组病毒在单个周期内复制速度快10倍，在多个复制周期中复制速度快1000倍，2)NS1A蛋白的这种突变导致病毒特异性RNA合成的速率显著增加，3)最近的蛋白质序列分析将NS1A的核输出信号置于上述毒力区域，以及4)NS1AL77A突变使病毒在没有事先适应的情况下对病毒产生更强的致病性(这一特征以前仅归因于病毒血凝素)，表明NS1a蛋白的这一特定区域显著影响病毒的毒力。在这些初步观察的基础上，了解NS1A蛋白中的这个功能位点(以下称为NS1A毒力区域或NS1AVR)及其影响毒力的机制是这一提议的重点。具体目的是：1.通过突变无毒株和高致病株的NS1a蛋白，确定围绕氨基酸L77的NS1AVR的边界。2.确定NS1AVR突变对NS1a蛋白核质穿梭的影响。3.用培养细胞和小鼠动物模型比较wt和NS1AVR突变病毒，通过分析感染组织的基因和/或细胞因子谱、病毒组织分布和复制率、中和抗体反应和/或白细胞渗透来确定毒力机制。

DESCRIPTION (provided by applicant): Influenza viruses (Orthomyxoviridae family) cause a highly contagious respiratory disease in humans resulting in approximately 36,000 deaths in the United States annually. The long term goal of this research is to reveal the mechanism by which the influenza non-structural protein-1 (NS1A) protein influences virus virulence utilizing in vitro, in vivo, and murine animal models. The hypothesis promoting this research is that the NS1A protein contains a previously uncharacterized site that participates in virus virulence. We propose that this region is a nuclear export signal (NES) and that inhibiting this NES enhances virulence of the virus by promoting NS1A nuclear functions. This hypothesis is based on observations that 1) an influenza A recombinant virus expressing an NS1 protein with a single alanine substitution at position L77 replicates 10-fold faster in a single cycle and 1000-fold faster in multiple cycles of replication, 2) this mutation in the NS1A protein leads to a substantially increased rate of virus-specific RNA synthesis, 3) recent protein sequence analysis places the nuclear export signal of NS1A in the virulence region identified above, and 4) the NS1A L77A mutation imparts increased pathogenicity to the virus in mice without prior adaptation (a characteristic previously attributed only to the viral hemagglutinin), demonstrating that this specific region of the NS1A protein significantly impacts the virulence of the virus. Based on these preliminary observations, the understanding of this functional site in the NS1A protein (hereafter termed the NS1A virulence region or NS1A VR) and the mechanism by which it influences virulence are the focal points of this proposal. The specific aims are to: 1. Define the boundaries of the NS1A VR that surround amino acid L77, by mutating the NS1A proteins of both avirulent and highly pathogenic strains. 2. Determine the effect of NS1A VR mutations on the nuclear-cytoplasmic shuttling of the NS1A protein. 3. Determine the mechanism of virulence by analyzing the gene and/or cytokine profile of infected tissues, virus tissue distribution and rate of replication, neutralizing antibody response, and/or leukocyte infiltration by comparing the wt and NS1A VR mutant viruses using cultured cells and a mouse animal model.