Autophagy: A Novel Antiviral Host Defense Pathway

描述（由申请人提供）：自噬过程或通过进化上保守的自噬体-溶酶体途径大量降解细胞蛋白的过程，对于营养胁迫、分化和发育以及负生长控制期间的存活非常重要。然而，几乎没有人知道这个细胞通路在防御细胞内病原体中的作用。本提案的主要目的是评估新的假设，即自噬作为细胞内抗病毒途径发挥作用，降解病毒颗粒，并被疾病发病机制所需的病毒基因产物拮抗。为了支持这一假设，我们以前已经证明，第一个确定的哺乳动物自噬基因，beclin 1，抑制辛德毕斯病毒复制，并保护小鼠免受致命的辛德毕斯病毒脑炎。此外，我们最近的数据表明，充分表征的IFN诱导型抗病毒分子PKR是单纯疱疹病毒（HSV）诱导的自噬所需的，并且HSV-1编码的神经毒力基因产物ICP 34.5拮抗哺乳动物细胞中的PKR依赖性自噬和酵母中的Beclin 1依赖性自噬。总之，这些发现使我们假设Beclin 1依赖性自噬在抗病毒宿主防御中的作用以及Beclin 1依赖性自噬在神经毒力中的ICP 34.5拮抗作用。在这个计划中，有四个具体的目的来研究相关的子假设，包括：（1）Beclin 1自噬基因是HSV-1诱导的自噬所必需的;（2）HSV-1 ICP34.5通过与Beclin 1自噬蛋白直接相互作用来拮抗病毒感染细胞中的自噬;（3）Beclin 1依赖的自噬通过促进胞质内病毒颗粒的降解而作为抗病毒宿主防御途径发挥作用，并且Beclin 1的这种功能被ICP 34.5拮抗;和（4）HSV ICP34.5通过涉及Beclin 1依赖性自噬的拮抗作用的机制调节HSV-1神经毒力。为了实现这些目标，我们将使用一种方法，该方法涉及联合使用自噬和Beclin 1蛋白表达缺陷的宿主细胞和编码ICP 34.5突变蛋白的重组HSV-1病毒，所述ICP34.5突变蛋白缺乏Beclin 1依赖性自噬的拮抗作用。我们将使用这些试剂来研究Beclin 1依赖性自噬在调节病毒复制中的作用，以及Beclin 1依赖性自噬的ICP 34.5拮抗作用在调节病毒神经毒力中的作用。总之，这些研究将提供关于自噬在抗病毒宿主防御中的作用以及病毒逃避自噬在神经毒力中的作用的新见解。

DESCRIPTION (provided by applicant): The process of autophagy, or bulk degradation of cellular proteins through an evolutionarily conserved autophagosomic-lysosomal pathway, is important in survival during nutrient stress, differentiation and development, and negative growth control. However, almost nothing is known about the role of this cellular pathway in defense against intracellular pathogens. The broad objective of this proposal is to evaluate the novel hypothesis that autophagy functions as an intracellular antiviral pathway that degrades viral particles and is antagonized by viral gene products required for disease pathogenesis. In support of this hypothesis, we have previously shown that the first identified mammalian autophagy gene, beclin 1, inhibits Sindbis virus replication and protects mice against lethal Sindbis virus encephalitis. In addition, our recent data demonstrate that the well-characterized IFN-inducible antiviral molecule, PKR, is required for herpes simplex virus (HSV)-induced autophagy and that the HSV-l-encoded neurovirulence gene product, ICP34.5, antagonizes PKR-dependent autophagy in mammalian cells and Beclin 1-dependent autophagy in yeast. Together, these findings lead us to hypothesize a role for Beclin 1-dependent autophagy in antiviral host defense and a role for ICP34.5 antagonism of Beclin 1-dependent autophagy in neurovirulence. In this proposal, there are four specific aims that investigate related sub-hypotheses, including: (1) the beclin 1 autophagy gene is required for HSV-l-induced autophagy; (2) HSV-1 ICP34.5 antagonizes autophagy in virally-infected cells through a direct interaction with the Beclin 1 autophagy protein; (3) Beclin 1-dependent autophagy functions as an antiviral host defense pathway by facilitating the degradation of intracytoplasmic viral particles and this function of Beclin 1 is antagonized by ICP34.5; and (4) HSV ICP34.5 regulates HSV-1 neurovirulence through a mechanism that involves antagonism of Beclin 1-dependent autophagy. To accomplish these aims, we will use an approach involving the combined use of host cells that are deficient in autophagy and expression of Beclin 1 protein and recombinant HSV-1 viruses that encode ICP34.5 mutant proteins that are deficient in antagonism of Beclin 1-dependent autophagy. We will use these reagents to investigate the role of Beclin 1-dependent autophagy in regulating viral replication and the role of ICP34.5 antagonism of Beclin 1-dependent autophagy in regulating viral neurovirulence. Together, these studies will provide novel insights about the role of autophagy in antiviral host defense and the role of viral evasion of autophagy in neurovirulence.