Studies in Poxvirus Host Range Genes and Tropism

描述(由申请人提供)：我们建议研究调节一种特定痘病毒的物种特异性和嗜性的分子途径，即粘液瘤病毒(MV)。MV是一种兔特有的痘病毒，它根据具体的兔子物种而引起明显不同的疾病，但对包括人类在内的所有其他宿主物种都是非致病的。我们的实验室广泛研究了MV及其在兔身上引起的疾病，称为粘液瘤病，作为一个模型系统来研究痘病毒发病的基本原理，特别是通过利用我们大量且不断增长的靶向MV基因敲除构建体。几年前，我们出人意料地发现，通常不允许MV感染的小鼠原代细胞，可以通过阻断细胞干扰素(干扰素)的反应而完全允许。这项工作随后发现，大多数接受测试的人类癌细胞对MV完全许可，MV在各种动物模型中作为治疗人类癌症异种移植的溶瘤疗法非常有效。我们还发现，两个病毒宿主范围因子(M-T5和M063)也是许多人类癌细胞中允许MV复制的关键因素，我们可以通过这些病毒蛋白及其宿主细胞蛋白靶标来操纵病毒的透过性和溶瘤作用。最后，我们最新的观察与这一提议有关，即干扰素和肿瘤坏死因子(TNF)协同保护原代人类细胞免受MV感染。我们建议：1-评估病毒宿主范围因素在人类癌细胞MV趋向性中的作用。我们建议通过突变、siRNA敲除、信号抑制剂和蛋白质微阵列来分析MV宿主范围蛋白与宿主细胞信号分子(如Akt)的相互作用。我们将测试一些辅助策略，如用于人类癌症化疗的信号调节药物，以增加更广泛范围的人类癌细胞的MV溶瘤潜力。2-探讨宿主干扰素和肿瘤坏死因子在MV趋向性中的作用。我们将研究几个关键的病毒宿主范围基因缺失的MV敲除病毒，以探讨它们在抑制干扰素或肿瘤坏死因子反应、调节MV趋向性和溶瘤作用中的作用。这一新信息将允许更合理的方法来优化针对更广泛范围的人类癌症的MV病毒治疗，并控制MV在原代非癌症人类细胞中的复制。公共卫生相关性：此前，我们对痘病毒致病机制的研究更多地集中在研究一种特定的兔痘病毒-粘液瘤病毒(MV)在兔宿主中引起疾病的基本机制。最近，我们发现MV还可以感染和杀死广泛的人类癌细胞，我们现在已经在动物模型中成功地使用MV治疗了几种类型的癌症。在这项计划中，我们将研究两个关键的病毒宿主范围因子及其细胞信号通路靶点，以帮助MV作为一种新的人类肿瘤溶解治疗的临床前开发。

DESCRIPTION (provided by applicant): We propose to examine the molecular pathways that mediate the species specificity and tropism of one particular poxvirus, namely myxoma virus (MV). MV is a rabbit-specific poxvirus that induces distinctly different disease profiles depending on the specific rabbit species but is nonpathogenic for every other host species, including man. Our lab has extensively studied MV and the disease it causes in rabbits, called myxomatosis, as a model system to investigate the fundamental principles of poxvirus pathogenesis, particularly by exploiting our large and growing collection of targeted MV gene knockout constructs. Several years ago, we unexpectedly discovered that primary mouse cells, which are normally nonpermissive for MV infection, could be rendered fully permissive by interrupting the cellular interferon (IFN) responses. This work then led to the discovery that the majority of human cancer cells tested were fully permissive for MV and that MV is remarkably effective as oncolytic therapy for the treatment of human cancer xenografts in a variety of animal models. We also discovered that two viral host range factors (M-T5 and M063) were also critical for permissive MV replication in many human cancer cells and we could manipulate viral permissiveness and oncolysis through these viral proteins and their host cell protein targets. Finally, our most recent observation related to this proposal is that primary human cells are protected from MV infection synergistically by IFN and tumor necrosis factor (TNF). We propose to: 1- Evaluate the roles of viral host range factors in MV tropism for human cancer cells. We propose to analyze the interactions of MV host range proteins with host cell signaling molecules (like Akt) by mutagenesis, siRNA knockdowns, signaling inhibitors and protein microarrays. We will test a number of adjunct strategies, like signaling modifier drugs used for cancer chemotherapy in man, to increase MV oncolytic potential for a wider spectrum of human cancer cells. 2- Investigate the role of host IFN and TNF responses in MV tropism. We will study MV knockout viruses that are deleted in several key viral host range genes to investigate their roles in inhibiting IFN or TNF responses, and modulating MV tropism and oncolysis. This new information will allow for more rational approaches to optimizing MV virotherapy against a wider spectrum of human cancers, and for controlling MV replication in primary noncancerous human cells. PUBLIC HEALTH RELEVANCE: Previously, our studies on poxvirus pathogenesis were more focused on studying the basic mechanisms by which one particular rabbit-specific poxvirus called myxoma virus (MV) causes disease in the rabbit host. Recently, we discovered that MV also infects and kills a wide spectrum of human cancer cells and we have now used MV to successfully treat several types of cancers in animal models. In this proposal, we will study two key viral host range factors and their cellular signaling pathway targets in order to assist the preclinical development of MV as a new oncolytic therapeutic for cancer in man.