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）在人类中引起高度传染性呼吸道疾病，导致美国每年约36,000人死亡。本研究的长期目标是利用体外、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。促进这项研究的假设是NS1A蛋白含有一个以前未被表征的参与病毒毒力的位点。我们认为该区域是核输出信号（NES），抑制该NES可通过促进NS1A核功能来增强病毒的毒力。这一假设是基于以下观察结果：1)表达L77位单丙氨酸取代的NS1蛋白的甲型流感重组病毒在单周期复制速度快10倍，在多周期复制速度快1000倍；2)NS1A蛋白的这种突变导致病毒特异性RNA合成速度大幅增加；3)最近的蛋白质序列分析将NS1A的核输出信号置于上述鉴定的毒力区。4) NS1A L77A突变在没有事先适应的情况下增加了病毒在小鼠中的致病性（这一特征以前只归因于病毒血凝素），表明NS1A蛋白的这一特定区域显著影响病毒的毒力。基于这些初步观察，了解NS1A蛋白中的这个功能位点（以下称为NS1A毒力区或NS1A VR）及其影响毒力的机制是本提案的重点。具体目标是：1。通过突变无毒和高致病性菌株的NS1A蛋白，确定氨基酸L77周围NS1A VR的边界。2. 确定NS1A VR突变对NS1A蛋白核胞质穿梭的影响。3. 利用培养细胞和小鼠动物模型比较wt和NS1A VR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/或白细胞浸润来确定毒力机制。

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.