Cellular DNAJ Proteins and SV40 Infection

这个由DiMaio博士领导的新项目是基于当前项目2中的一个发现。 融资期。多瘤病毒BK病毒和JC病毒可引起免疫抑制的严重疾病， 包括癌症患者在内的个体，并且像它们的近亲SV 40一样，是推定的人类肿瘤病毒。 在该基金的支持下，我们发现细胞辅伴侣DNAJ-B12和DNAJ-B14是 这三种病毒的有效感染所需的条件。当这些基因的表达被抑制时， shRNAs，主要的早期蛋白，大T抗原的表达有实质性的减少。病毒 与细胞表面的结合似乎未受损害，表明DNAJ-B12/14敏感步骤在某些情况下是 病毒进入、细胞内运输或脱壳方面。我们将进行一系列的生化，细胞 生物学和遗传学研究，以阐明DNAJ-B12/14在SV 40感染中发挥的机制作用。 我们将确定在缺乏DNAJ-B12/14功能的细胞中感染进展的程度，并确定 病毒进入/贩运/去包被过程中的步骤被阻止。我们将进行突变和生化 分析DNAJ-B12/14以确定其在分子水平上的作用模式。病毒逃逸突变体， 尽管DNAJ-B12/14抑制仍允许感染，将被分离和表征。最后，我们将使用 利用shRNA技术确定DNAJ基因家族的其他成员是否是感染所必需的 由多瘤病毒和其他病毒，包括EB病毒与米勒博士合作。这些 实验将为肿瘤病毒感染的过程提供新的见解，并描述肿瘤病毒在肿瘤中的作用。 新的抗病毒靶点

This new project headed by Dr. DiMaio is based on a discovery made in project 2 during the current funding period. The polyomaviruses, BK virus andJC virus, cause serious diseases in immunosuppressed individuals including cancer patients and, like their close relative SV40, are putative human tumor viruses. With the support of this grant, we discovered that the cellular co-chaperones DNAJ-B12 and DNAJ-B14 are required for efficient infection by these three viruses. When expression of these genes is repressed by shRNAs, there is a substantial reduction in expression of the major early protein, large T antigen. Virus binding to the cell surface appears unimpaired, suggesting that the DNAJ-B12/14 sensitive step(s) is in some aspect of virus entry, intracellular trafficking, or uncoating. We will conduct a series of biochemical, cell biological and genetic studies to elucidate the mechanistic role played by DNAJ-B12/14 in SV40 infection. We will determine how far infection proceeds in cells lacking DNAJ-B12/14 function, and determine what step in the virus entry/trafficking/uncoating process is blocked. We will conduct mutational and biochemical analysis of DNAJ-B12/14 to determine its mode of action at the molecular level. Viral escape mutants that allow infection despite DNAJ-B12/14 repression will be isolated and characterized. Finally, we will use shRNA technology to determine whether other members of the DNAJ gene family are required for infection by the polyomaviruses and other viruses, including Epstein-Barr virus in collaboration with Dr. Miller. These experiments will provide new insights into the process of tumor virus infection and characterize the role of new putative anti-viral targets.