Studies in Poxvirus Host Range Genes and Tropism

描述（由申请人提供）：我们建议研究介导一种特定痘病毒，即黏液瘤病毒（MV）的物种特异性和趋向性的分子途径。MV是一种兔特异性痘病毒，根据特定的兔种诱导明显不同的疾病特征，但对所有其他宿主物种（包括人类）都无致病性。我们的实验室广泛研究了水痘病毒及其在兔子中引起的称为粘液瘤病的疾病，作为研究水痘病毒发病的基本原理的模型系统，特别是通过利用我们大量和不断增长的靶向水痘病毒基因敲除构建体。几年前，我们意外地发现，正常情况下对MV感染不允许的小鼠原代细胞，可以通过中断细胞干扰素（IFN）反应而变得完全允许。这项工作随后发现，大多数被测试的人类癌细胞是完全允许MV的，并且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）在许多人类癌细胞中也对容许性MV复制至关重要，我们可以通过这些病毒蛋白及其宿主细胞蛋白靶点来操纵病毒的容许性和溶瘤。最后，我们最近与这一建议相关的观察结果是，IFN和肿瘤坏死因子（TNF）协同保护人类原代细胞免受MV感染。我们建议：1-评估病毒宿主范围因子在人癌细胞趋向性中的作用。我们建议通过诱变、siRNA敲低、信号抑制剂和蛋白质微阵列分析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修饰剂药物，以增加更广泛的人类癌细胞的MV溶瘤潜力。2-研究宿主IFN和TNF在MV趋向性中的作用。我们将研究在几个关键病毒宿主范围基因中缺失的MV敲除病毒，以研究它们在抑制IFN或TNF反应、调节MV向性和溶瘤中的作用。这一新信息将允许更合理的方法来优化针对更广泛的人类癌症的MV病毒治疗，并控制原发非癌人类细胞中的MV复制。公共卫生相关性：以前，我们对痘病毒发病机制的研究更多地集中在研究一种称为黏液瘤病毒（MV）的兔特异性痘病毒在兔宿主中引起疾病的基本机制。最近，我们发现MV也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。在本研究中，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作为一种新的人类癌症溶瘤治疗药物的临床前开发。

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.