GENE REGULATION OF MAMMALIAN DNA VIRUSES

描述：（改编自研究者摘要）：申请人的 长期的兴趣是确定特定痘病毒基因在 发病机制他们在此集中研究痘病毒编码的蛋白酶抑制剂 （serpins）。正痘病毒，包括牛痘病毒（CPV），编码三种丝氨酸蛋白酶抑制剂： SPI-1、SPI-2/crmA和SPI-3。兔痘病毒粘液瘤病毒（MYX）编码两个 功能性丝氨酸蛋白酶抑制剂：SERP 1和SERP 2。丝氨酸蛋白酶抑制剂的特异性主要取决于 通过反应位点环（RSL）内P1残基的身份， 与靶蛋白酶相互作用。第一个具体目标是确定 Serpin对在感染兔子时是否在功能上可互换 和鸡胚绒毛尿囊膜。CPV crmA和MYX SERP 2都具有 在P1位置的Asp，并抑制类似的蛋白酶（半胱天冬酶和颗粒酶 B）体外。初步研究表明，毒力和控制的主要 病变发展可能是SERP 2的独立功能。定点突变体 影响crmA和SERP 2的P1残基，将用于确定 这些丝氨酸蛋白酶抑制剂的功能不依赖于蛋白酶抑制。 其中SERP 2或crmA被非丝氨酸蛋白酶抑制剂杆状病毒p35替代的实验， 半胱天冬酶抑制剂，将评估颗粒酶B在发病机制中的作用 和病变发展。MYX SERP 1和CPV SPI-3各自具有P1 Arg残基，并且 非常相似的体外蛋白酶抑制特性。替代SERP 1 MYX与SPI-3将SPI-3（通常为CPV细胞内蛋白）改变为 分泌蛋白我们将比较的动力学和生物学性质的 SPI-3和SERP 1蛋白，以研究为什么基因在功能上不 可互换，以及为什么MYX中的SPI-3被分泌。具体目标2关注 SPI-1，兔痘病毒在整个宿主范围内所需。他们将 表征SPI-1的独特细胞内修饰，其依赖于 功能性P1残基，并且其可以代表 丝氨酸蛋白酶抑制剂-蛋白酶复合物他们设计了一种基因筛选方法， 和SPI-1 P14突变的基因外抑制子， SPI-1的结构和功能。具体目标3提出免疫共沉淀 和交联研究作为鉴定靶蛋白酶的手段， 痘病毒丝氨酸蛋白酶抑制剂他们还提出了相互作用蛋白质的遗传筛选 基于酵母双杂交系统，以及基于涉及 噬菌体和λ阻遏物。他们的研究旨在提供一个 通过详细描述痘病毒丝氨酸蛋白酶抑制剂的相互作用， 蛋白酶和其他细胞和病毒蛋白质。

DESCRIPTION: (Adapted from the Investigator's abstract): The applicant's long-term interests are in determining the roles of specific poxvirus genes in pathogenesis. They focus here on poxvirus-encoded proteinase inhibitors (serpins). Orthopoxviruses, including cowpox virus (CPV), encode three serpins: SPI-1, SPI-2/crmA and SPI-3. The leporipoxvirus myxoma virus (MYX) encodes two functional serpins: SERP1 and SERP2. Serpin specificity is determined largely by the identity of the P1 residue within the Reactive Site Loop (RSL) which interacts with the target proteinase. The first specific aim is to determine whether serpin pairs are functionally interchangeable in infection of rabbits and of the chicken chorioallantoic membrane. CPV crmA and MYX SERP2 both have Asp at the P1 position, and inhibit similar proteinases (caspases and granzyme B) in vitro. Preliminary studies suggest that virulence and control of primary lesion development may be separable functions of SERP2. Site-directed mutants affecting the P1 residue of crmA and SERP2 will be used to determine if any of the functions of these serpins are independent of proteinase inhibition. Experiments where SERP2 or crmA is replaced by the non-serpin baculovirus p35, an inhibitor of caspases, will evaluate the role of granzyme B in pathogenesis and lesion development. MYX SERP1 and CPV SPI-3 each have a P1 Arg residue, and very similar proteinase inhibitory properties in vitro. Replacement of SERP1 in MYX with SPI-3 changes SPI-3, normally a CPV intracellular protein, into a secreted protein. We will compare the kinetic and biological properties of SPI-3 and SERP1 proteins to investigate why the genes are not functionally interchangeable, and why SPI-3 in MYX is secreted. Specific aim 2 concerns SPI-1, which is required in rabbitpox virus for full host range. They will characterize a unique intracellular modification of SPI-1 that is dependent on a functional P1 residue, and which may represent the vestige of a serpin-proteinase complex. They have devised a genetic screen for intragenic and extragenic suppressors of a SPI-1 P14 mutation that will give information on SPI-1 structure and function. Specific aim 3 proposes coimmunoprecipitation and cross-linking studies as a means of identifying target proteinases for poxvirus serpins. They also propose genetic screens for interacting proteins based on the yeast 2-hybrid system, and on a bacterial system involving the phage and lambda repressor. Their studies are intended to provide an understanding of how poxvirus serpins function by detailing their interactions with proteinases and with other cellular and viral proteins.