GENE REGULATION OF MAMMALIAN DNA VIRUSES

我们的长期兴趣是识别相关的特征性基因 在痘病毒的发病机制中。我们建议重点关注三个基因产物 兔痘病毒(RPV)：PS/hr(B5R ORF)、SPI和SPI-2。这些毒力 基因驻留在病毒基因组的末端区域，并且 与控制蛋白(PS/hr)或丝氨酸同源 蛋白水解酶抑制剂(SPI-1和-2)。野生型RPV毒力很强， 在体外生长在广泛的细胞系上，并产生红色、出血性 鸡胚绒毛尿囊膜(CAM)上的麻点。 在动物模型系统(PS/hr)中，所有三个基因中的突变都被减弱 和SPI-2)，或显示主机范围限制(PS/小时和SPI-1)，或生成 CAM上含有炎性细胞的白色麻点(PS/小时，SPI-1和- 2)。PS/hr基因与补体的H因子具有同源性 途径和凝血途径的XIII因子，并编码45 细胞外膜病毒中发现的KDA糖蛋白和一种 可溶性35 kDa形式，由独立于病毒的过程分泌 成熟。我们计划构建病毒来合成分泌的 或膜相关形式，以便识别哪种形式的 PS/HR蛋白负责控制炎症，这是 全主机范围所需。然后将使用定点突变 以确定蛋白质的关键结构域。基于生物化学的检测方法 PS/hr蛋白所显示的同源性将被用来确定 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，并将被检测为抑制 白介素1β转换酶(ICE)的已知活性 牛痘病毒(CPV)SPI-2同源物。RPV SPI-1蛋白是必需的 在A549细胞上形成斑块，而该基因产物的缺乏导致 诱导细胞凋亡。该主机范围限制的机制将 有待进一步分析。为了探索可能的职能重叠 在SPI-1和SPI-2基因之间，大约45%相同，RPV 缺乏这两种基因的突变体将被构建和测试。不同的 RPV和CPV中单一SPI基因中断的影响将通过 基因交换。我们计划筛选过表达的RPV SPI-1和-2和 Swinpox丝氨酸蛋白抑制特定丝氨酸蛋白酶的研究 体外，并抑制参与CTL-和NK-的丝氨酸蛋白酶。 介导的靶细胞的细胞溶解。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，以及 通过使用标记的蛇类进行生物化学。这些化验结果将 与对击倒和场地行为的广泛分析有关- 在鸡蛋、小鼠和兔子中定向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.