E3L AND POXVIRUS REPLICATION/INTERFERON RESISTANCE

干扰素系统是脊椎动物的主要防御系统之一 对抗病毒感染一些抗干扰素病毒最近 被定性。拟议研究的长期目标是 了解病毒对干扰素产生抗性的机制。 具体的目的是了解痘病毒之一的作用， 干扰素系统的抑制剂在这些病毒的复制和它们的 抗干扰素。自从痘病毒被开发为载体 对于人类疫苗，可以拯救其他病毒免受IFN的影响， 因为C组轮状病毒，在人类中引起严重腹泻，编码 类似的PKR抑制剂，这项研究可能会影响健康问题。 牛痘病毒在病毒复制中的作用及对干扰素的抗性 IFN系统的抑制剂将通过使用突变菌株来评价 病毒vP 1080，已经删除了与抗性有关的基因， IFN，E3 L基因。该基因也与抑制 在牛痘病毒感染的细胞凋亡，并可以转化细胞， 文化在vp 1080中IFN系统的各种组分的活化- 将对感染的细胞进行分析。鼠痘病毒（另一种痘病毒） 将制备缺失E3 L同源物的基因，并研究其发病机制。 改造后的病毒将在小鼠中进行试验。结构与功能的关系 对于由E3 L编码的蛋白质，将确定p25。p25特异性 结合双链（ds）RNA，并与其他几种 dsRNA结合蛋白，包括IFN诱导的蛋白激酶， 结合HIV反式激活（tar）位点TRBP的人蛋白，和 轮状病毒NSP 3蛋白。 结构/功能关系 保守的dsRNA结合结构域将通过定点酶切来确定。 诱变带有该结构域的蛋白质的晶体结构将被 测定这项工作可能会为蛋白质如何识别 dsRNA。其他牛痘病毒基因可能影响病毒对 IFN将通过选择可以允许在细胞中生长的基因来分离。 缺失E3 L基因的痘苗病毒的IFN的存在。这项工作将 提供痘病毒如何进化出对干扰素的抗性的洞察。

The interferon (IFN) system is one of the primary vertebrate defenses against virus-infection. A number of IFN-resistant virus have recently been characterized. The long-term objectives of the proposed research are to understand the mechanisms involved in resistance of viruses to IFN. The specific aims are to understand the role of one of the poxvirus inhibitors of the IFN system in replication of these viruses and in their resistance to IFN. Since poxviruses, which are being developed as vectors for human vaccines, can rescue other viruses from the effects of IFN, and since group C rotaviruses, which cause severe diarrhea in humans, encode a similar PKR inhibitor, this research may impact health issues. The role in virus replication and resistance to IFN of the vaccinia virus inhibitor of the IFN system will be evaluated by use of a mutant strain of virus, vPl080, that has been deleted for a gene involved in resistance IFN, the E3L gene. This gene has also been implicated in suppression of apoptosis in vaccinia virus infected cells, and can transform cells in culture. Activation of various components of the IFN system in vp1080- infected cells will be analyzed. Ectromelia virus (another poxvirus) deleted for the E3L homologue will be prepared and pathogenesis of this altered virus will be tested in mice. Structure function relationships for the protein encoded by E3L, p25, will be determined. p25 specifically binds double-stranded (ds)RNA, and shares homology with several other dsRNA-binding proteins, including the IFN-induced protein kinase, the human protein that binds to the HIV transactivation (tar) site, TRBP, and the rotavirus NSP3 protein. Structure/function relationships in this conserved dsRNA-binding domain will be determined by site-directed mutagenesis. Crystal structures for proteins bearing this domain will be determined. This work may provide light into how proteins recognize dsRNA. Other vaccinia virus-genes that may affect viral sensitivity to IFN will be isolated, by selecting for genes that can allow growth in the presence of IFN of vaccinia virus deleted the E3L gene. This work will provide insight into how the poxviruses have evolved resistance to IFN.