Autophagy: A Novel Antiviral Host Defense Pathway

描述（由申请人提供）：自噬过程，或通过进化保守的自噬体-溶酶体途径对细胞蛋白的大量降解，在营养胁迫、分化和发育以及负生长控制期间的生存中是重要的。然而，关于这种细胞途径在防御细胞内病原体中的作用，我们几乎一无所知。本提案的主要目的是评估自噬作为细胞内抗病毒途径的新假设，该途径降解病毒颗粒并被疾病发病所需的病毒基因产物拮抗。为了支持这一假设，我们之前已经证明，第一个发现的哺乳动物自噬基因beclin 1可以抑制Sindbis病毒的复制，并保护小鼠免受致命的Sindbis病毒脑炎的侵袭。此外，我们最近的数据表明，明确表征的ifn诱导的抗病毒分子PKR是单纯疱疹病毒（HSV）诱导的自噬所必需的，并且HSV-l编码的神经毒力基因产物ICP34.5可以拮抗哺乳动物细胞中依赖于PKR的自噬和酵母中依赖于Beclin 1的自噬。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依赖性自噬的ICP34.5拮抗在神经毒力中的作用。在本研究中，有四个具体的目的来研究相关的子假设，包括：(1)hsv -l诱导的自噬需要beclin - 1基因；(2) HSV-1 ICP34.5通过与Beclin -1自噬蛋白的直接相互作用拮抗病毒感染细胞的自噬；(3) Beclin - 1依赖性自噬通过促进胞浆内病毒颗粒的降解而作为抗病毒宿主防御途径，Beclin - 1的这一功能可被ICP34.5拮抗；(4) HSV ICP34.5通过拮抗Beclin -1依赖性自噬的机制调控HSV-1的神经毒力。为了实现这些目标，我们将使用一种方法，包括结合使用自噬缺陷的宿主细胞和Beclin 1蛋白的表达，以及重组HSV-1病毒，这些病毒编码缺乏Beclin 1依赖性自噬拮抗的ICP34.5突变蛋白。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ICP34.5拮抗Beclin 1依赖性自噬在调节病毒神经毒力中的作用。总之，这些研究将为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提供新的见解。

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.