Viral Stress-inducible Gene Expression

描述(申请人提供)：哺乳动物细胞的病毒感染导致特定细胞基因表达模式的快速和深刻变化。由这些基因编码的蛋白质，即病毒应激诱导基因(VSIG)，负责维持感染细胞中的病毒稳态。许多VSIG也可以在未感染的细胞中由干扰素(IFN)或双链(DS)RNA诱导，这是病毒感染的常见副产品。在这里，我们建议确定响应I型干扰素、双链RNA和仙台病毒感染的细胞基因表达谱的变化模式。定制的cDNA微阵列将被用来测量病毒感染或干扰素/双链RNA处理细胞中相关细胞mRNAs的水平，并与未处理细胞中的相应水平进行比较。突变的细胞系，特别是在干扰素或dsRNA诱导的信号通路方面存在缺陷，将被用于评估这些通路在仙台病毒基因诱导中的作用。此外，我们建议描绘三种不同的信号通路，以诱导编码P56蛋白家族的VSIG的一个共同子集的转录。为此，我们将使用Jak-STAT、IRF3和NFkappaB信号通路的特定成分缺乏的突变细胞系，以及化学抑制剂和siRNA。DsRNA信号使用Toll样受体3来诱导P56家族基因的转录，最近我们发现受体酪氨酸磷酸化在这一过程中是必不可少的。我们建议确定TLR3的特定磷酸酪氨酸残基在介导dsRNA信号导致P56和其他基因的诱导中的作用。需要TLR3磷酸化的生化基础将通过检测表达Wt或突变的TLR3蛋白的细胞中相关转录因子和蛋白激酶的激活来研究。这些研究将阐明TLR和Jak-STAT信号通路之间的串扰模式，以便在病毒感染的细胞中进行适当的基因诱导。

DESCRIPTION (provided by applicant): Viral infection of mammalian cells causes rapid and profound changes in the expression patterns of specific cellular genes. The proteins encoded by these genes, the viral stress-inducible genes (VSIG), are responsible for maintaining viral homeostasis in the infected cells. Many of the VSIG can also be induced in uninfected cells by interferons (IFN) or double-stranded (ds) RNA, common byproducts of viral infection. Here, we propose to identify the patterns of changes in cellular gene expression profiles in response to type I IFN, dsRNA and Sendai virus infection. Customized cDNA microarrays will be used to measure the levels of relevant cellular mRNAs in virus-infected or IFN/dsRNA treated cells and compared with the corresponding levels in untreated cells. Mutant cell lines, specifically defective in IFN or dsRNA-elicited signaling pathways, will be used to assess the contributions of these pathways in gene induction by Sendai virus. Furthermore, we propose to delineate the distinct signaling pathways used by the three agents to induce transcription of a common subset of VSIG that encode the P56 family of proteins. For this purpose, we will use mutant cell lines deficient in specific components of the Jak-STAT, the IRF3 and the NFkappaB signaling pathways as well as chemical inhibitors and siRNAs. DsRNA signaling uses Toll-like receptor 3 for inducing transcription of the P56 family of genes and it has recently been shown by us that receptor tyrosine phosphorylation is essential for this process. We propose to identify the roles of specific phosphotyrosine residues of TLR3 in mediating dsRNA-signaling that leads to the induction of P56 and other genes. The biochemical basis of the need for TLR3 phosphorylation will be investigated by examining activation of the relevant transcription factors and protein kinases in cells expressing Wt or mutant TLR3 proteins. These studies will illuminate the modes of cross-talk between the TLR and the Jak-STAT signaling pathways for appropriate gene induction in virus-infected cells.