GENE REGULATION OF MAMMALIAN DNA VIRUSES

我们的长期兴趣是确定相关的特征基因 在痘病毒发病机制中的作用。 我们建议集中在三个基因产物， 兔痘病毒（RPV）：ps/hr（B5 R ORF）、SPI和SPI-2。 这些毒力 基因位于病毒基因组的末端区域， 与补体控制蛋白（ps/hr）或丝氨酸同源 蛋白酶抑制剂（SPI-1和-2）。 野生型RPV是高毒性的， 在体外广泛的细胞系上生长， 鸡胚绒毛尿囊膜上的麻点。 所有三个基因中的突变体在动物模型系统中减弱（ps/hr 和SPI-2），或显示寄主范围限制（ps/hr和SPI-1），或产生 CAM上含有炎性细胞的白色斑点（ps/hr，SPI-1和- 2）。 ps/hr基因既与补体H因子有同源性， 途径和凝血途径的因子XIII，并编码45 在细胞外包膜病毒（EEV）中发现的kDa糖蛋白和 可溶性35 kDa形式，其由不依赖于病毒的过程分泌 成熟 我们计划构建病毒， 或膜相关形式，以确定哪种形式的 ps/hr蛋白负责控制炎症， 需要完整的主机范围。 然后将使用定点诱变 来识别蛋白质的关键区域 生化分析基于 由PS/HR蛋白显示的同源性将用于确定 PS/HR蛋白抑制炎症。 RPV SPI-1和SPI-2蛋白 也调节鸡蛋中的炎症，并将测定对 白细胞介素-1 β转化酶（ICE），基于已知的活性， 牛痘病毒（CPV）SPI-2同系物。 需要RPV SPI-1蛋白 A549细胞上的空斑，缺乏这种基因产物会导致 诱导细胞凋亡。 此主机范围限制的机制将 进一步分析。 为了探索可能的职能重叠 在SPI-1和SPI-2基因之间，它们大约有45%的相同性， 将构建和测试缺乏这两种基因的突变体。 不同 RPV和CPV中单个SPI基因破坏的影响将通过 基因交换 我们计划筛选过表达的RPV SPI-1和SPI-2， 用于抑制确定的丝氨酸蛋白酶的猪痘丝氨酸蛋白酶抑制剂蛋白 体外，并用于抑制涉及CTL-和NK-的丝氨酸蛋白酶。 介导的靶细胞溶解。 与其他蛋白质的相互作用将 通过酵母双杂交系统进行遗传筛选， 通过使用标记的丝氨酸蛋白酶抑制剂进行生物化学处理。 这些分析的结果将 与敲除行为和位点的广泛分析有关- 鸡蛋、小鼠和兔子中的定向RPV突变体。

Our long term interest is in identifying the characterizing genes involved in poxvirus pathogenesis. We propose to focus on three gene products of rabbitpox virus (RPV): ps/hr (B5R ORF), SPI, and SPI-2. These virulence genes reside in the terminal regions of the viral genome, and have homology either to complement control proteins (ps/hr) or to serine proteinase inhibitors (SPI-1 and -2). Wild type RPV is highly virulent, grows on a wide range of cell lines in vitro, and gives red, hemorrhagic pocks on the chorioallantoic membrane (CAM) of embryonated chicken eggs. Mutants in all three genes are attenuated in anima model systems (ps/hr and SPI-2), or show host range restriction (ps/hr and SPI-1), or produce white pocks containing inflammatory cells on the CAM (ps/hr, SPI-1 and - 2). The ps/hr gene has homology both to factor H of the complement pathway and to factor XIII of the coagulation pathway, and encodes a 45 kDa glycoprotein found in extracellular enveloped virus (EEV) and a soluble 35 kDa form which is secreted by a process independent of virus maturation. We plan to construct viruses synthesizing either the secreted or membrane-associated form only in order to identify which form of the ps/hr protein is responsible for control of inflammation and which is needed for full host range. Site-directed mutagenesis will then be used to identify critical domains of the protein. Biochemical assays based on the homologies shown by the ps/hr protein will be used to determine how the ps/hr protein inhibits inflammation. The RPV SPI-1 and SPI-2 proteins also regulate inflammation in eggs, and will be assayed for inhibition of interleukin-1beta converting enzyme (ICE) based on the known activity of the cowpox virus (CPV) SPI-2 homolog. The RPV SPI-1 protein is required for plaquing on A549 cells, and lack of this gene product results in the induction of apoptosis. The mechanism of this host range restriction will be analyzed further. In order to explore possible functional overlap between the SPI-1 and SPI-2 genes, which are about 45% identical, an RPV mutant lacking both genes will be constructed and tested. The different effects of single SPI-gene disruptions in RPV and CPV will be examined by gene swapping. We plan to screen overexpressed RPV SPI-1 and -2 and swinepox serpin proteins for inhibition of defined serine proteinases in vitro, and for inhibition of serine proteinases involved in CTL- and NK- mediated cytolysis of target cells. Interactions with other proteins will be screened genetically by means of the yeast two-hybrid system, and biochemically by use of tagged serpins. The results of these assays will be linked to an extensive analysis of the behavior of knockout and site- directed RPV mutants in eggs, mice and rabbits.