GENE REGULATION OF MAMMALIAN DNA VIRUSES

描述：(改编自调查人员摘要)：申请人的 长期的兴趣是确定特定的痘病毒基因在 发病机制。他们在这里关注的是痘病毒编码的蛋白酶抑制剂 (蛇类)。正痘病毒，包括牛痘病毒(CPV)，编码三种蛇： SPI-1、SPI-2/CrmA和SPI-3。麻痘病毒粘液瘤病毒(MYX)编码两种病毒 功能丝氨酸：SERP1和SERP2。Serpin的特异性在很大程度上是由 通过反应位点环(RSL)内的P1残基的同一性，该反应位点环(RSL) 与目标蛋白酶相互作用。第一个具体目标是确定 在兔感染中，蛇链对是否具有互换功能 和鸡绒毛膜尿囊膜。CPV CrmA和MYX SERP2都有 位于P1位的天冬氨酸氨基转移酶，抑制类似的蛋白酶(半胱氨酸天冬氨酸氨基转移酶和颗粒酶 B)体外培养。初步研究表明，原生生物的毒力和控制 病变的发生可能是SERP2的独立功能。定点突变体 影响CrmA和SERP2的P1残基将被用来确定是否有 这些蛇类的功能不受蛋白酶抑制的影响。 SERP2或CrmA被非丝氨酸杆状病毒P35取代的实验， 半胱氨酸氨基转移酶的抑制剂，将评估颗粒酶B在发病机制中的作用 和病变的发展。Myx SERP1和CPV SPI-3各自具有P1 Arg残基，并且 在体外非常相似的蛋白水解酶抑制特性。中SERP1的替换 带有Spi-3的Myx将Spi-3，通常是一种CPV细胞内蛋白，改变为 分泌的蛋白质。我们将比较它们的动力学和生物学特性 SPI-3和SERP1蛋白来研究为什么这些基因没有功能 可互换，以及为什么MYX中的SPI-3被分泌。具体目标2关注点 SPI-1，是兔痘病毒在整个宿主范围内所必需的。他们会 表征SPI-1的一种独特的细胞内修饰，它依赖于 一个功能的P1残基，它可能代表一个 丝氨酸-蛋白酶复合体。他们设计了一种基因内筛查 和SPI-1 P14突变的基因外抑制物，将提供信息 关于SPI-1的结构和功能。具体目标3提出免疫共沉淀 以及作为识别靶蛋白的一种手段的交联性研究 痘病毒毒蛇。他们还建议对相互作用的蛋白质进行基因筛选。 基于酵母双杂交系统，以及涉及 噬菌体和波长抑制因子。他们的研究旨在提供一种 通过详细描述它们之间的相互作用来理解痘病毒蛇毒的功能 与蛋白酶以及其他细胞和病毒蛋白结合。

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.