Bik Promotes Cleavage of Viral Proteins to Enhance Influenza A Virus Infection

 描述（由申请人提供）：流感（流感）是一种高度传染性的呼吸道疾病，每年影响数百万人，给经济带来重大损失。目前，甲型流感病毒（IAV）亚型H1N1是季节性流行病的原因，可能导致严重的呼吸道疾病和死亡。据估计，每年的流感病毒流行使美国损失了104亿美元的直接医疗费用和164亿美元的潜在收入损失。新的流感病毒株的频繁出现使有效疫苗的生产变得困难。因此，需要研究IAV复制和传播的详细分子机制，以设计有效的抗病毒药物和疫苗。流感诱导受感染细胞中的细胞死亡以促进病毒复制。我们的初步研究确定了Bcl-2相互作用的杀伤细胞（Bik）作为一个重要的细胞蛋白，促进IAV感染的人气道上皮细胞（AEC）和小鼠。我们发现，与bik +/+细胞相比，ik-/-细胞中的IAV复制减弱，如病毒滴度降低所示。感染后，bik缺陷型小鼠气道上皮细胞（MAEC）在感染后表现出更稳定的跨上皮抗性，对感染诱导的细胞死亡不太敏感， 与bik充足的MAEC相比，病毒RNA水平降低。Bik的敲低降低了病毒蛋白表达水平和人气道上皮细胞（HAECs）对IAV诱导的细胞死亡的敏感性。对感染IAV的小鼠的存活分析表明，bik-/-小鼠在感染后存活的天数明显更长，并且与bik +/+小鼠相比，死于感染的可能性低10倍。此外，与bik-/-小鼠相比，bik +/+小鼠体重减轻明显更多。IAV以Bik依赖的方式激活caspase 3，当Bik被敲低时，病毒NP和M2蛋白的切割被抑制，这意味着Bik介导的病毒蛋白的caspase切割与病毒复制有关。因此，我们假设Bik对于病毒蛋白组分的细胞内切割以促进病毒复制至关重要。尽管病毒蛋白的切割已显示促进病毒感染性，但对相关宿主细胞蛋白酶知之甚少。本研究将探讨半胱天冬酶在增强病毒感染性中的作用，并测试是否靶向抑制半胱天冬酶减轻IAV感染性。这一假设将在具体目标1和2中得到检验。目的1研究Bik是否介导IAV感染细胞中病毒蛋白的半胱天冬酶依赖性切割。目的2将确定调节Bik的表达或抑制半胱天冬酶激活是否减轻病毒复制和对IAV诱导的体内死亡率的易感性。更好地理解IAV如何操纵宿主细胞蛋白以促进病毒复制的分子机制将提供对靶向细胞和/或病毒蛋白以减少人类感染和疾病进展的新治疗方法的深入了解。

 DESCRIPTION (provided by applicant): Influenza (flu) is a highly contagious respiratory disease that affects millions of people each year with significant cost to the economy. Currently, influenza A virus (IAV) of the subtype H1N1 is responsible for seasonal epidemics that may result in severe respiratory illness and deaths worldwide. The annual Influenza virus epidemics are estimated to cost the U.S. 10.4 billion in direct medical expenses and 16.4 billion in lost potential earnings. The frequent appearances of new strains of flu made the generation of effective vaccines difficult. Therefore, the detailed molecular mechanisms how IAV replicates and propagates need to be investigated to design effective antiviral drugs and vaccines. Influenza induces cell death in infected cells to facilitate virus replication. Our preliminary studies identified the Bcl-2 interacting killer (Bik) as a crucial cellular protein promoting IAV infection in human airway epithelial cells (AECs) and in mice. We found that IAV replication was attenuated in bik-/- compared to bik+/+ cells, as indicated by reduced viral titers. Following infection, bik-deficient mouse airway epithelial cells (MAECs) showed more stable trans-epithelial resistance following infection, were less sensitive to infection-induced cell death, and had reduced levels of viral RNA compared to bik-sufficient MAECs. Knockdown of Bik reduced viral protein expression levels and the sensitivity of human airway epithelial cells (HAECs) to IAV-induced cell death. Survival analysis of mice infected with IAV showed that bik-/- mice survived for significantly longer days after infection and were 10-fold less likely to die from infection compared to bik+/+ mice. Additionally, the bik+/+ mice lost significantly more weight compared to the bik-/- mice. IAV activated caspase 3 in a Bik-dependent manner and cleavage of viral NP and M2 proteins were inhibited when Bik was knocked down, implying that Bik-mediated caspase cleavage of viral proteins is linked to virus replication. Thus, we hypothesize that Bik is crucial for intracellular cleavage of components of viral protein(s) to promote virus replication. Although cleavage of viral proteins has been shown to promote virus infectivity, littl is known about relevant host cell proteases. This study will investigate the role of caspases in enhancing virus infectivity and tests whether targeted inhibition of caspases mitigate IAV infectivity. This hypothesis will be tested in Specific Aims 1 and 2. Aim 1 will investigate whethe Bik mediates caspase-dependent cleavage of viral proteins in IAV infected cells. Aim 2 will determine whether modulating the expression of Bik or inhibition of caspase activations mitigate viral replication and susceptibility to IAV- induced mortality in vivo. A better understanding of te molecular mechanisms how IAV manipulates host cellular proteins to facilitate virus replication will provide insight into new therapeutic approaches that target cellular and/or viral proteins to reduce infection and disease progression in humans.