Rotavirus: Studies of Intestinal Tropism, Viral Entry, and Rescue

描述(由申请人提供)： 轮状病毒(RV)是全球婴幼儿严重腹泻的最重要原因。这些病毒也是健康成年人、老年人和免疫功能低下者患病的常见原因。事实上，所有哺乳动物都能有效地感染自己的同源宿主RV，但不能感染异源RV。最近，有效的减毒活轮状病毒疫苗已经上市，但其作用机制和减毒的分子基础尚不清楚。轮状病毒感染主要局限于小肠的绒毛上皮。值得注意的是，肠道细胞中异源RV复制的宿主范围限制的基础仍不清楚。轮状病毒由于其相对较小的基因组大小、宿主范围和组织嗜性，是研究宿主上皮细胞与肠道病原体相互作用的高度易操作的模型系统。轮状病毒也是靶向人类小肠上皮细胞的潜在载体，也是评估针对肠道病原体的疫苗接种策略的理想系统。我们的目标是继续研究轮状病毒与宿主的相互作用，以更好地了解肠道微生物的发病机制。该建议的具体目的是：1)在小鼠模型中，使用病毒重组体表征选定的同源和异种RV的宿主限制性肠道细胞趋向性的遗传基础。我们的假设是，介导细胞进入的轮状病毒表面蛋白之一(VP4)和阻断宿主干扰素反应的轮状病毒IRF3拮抗剂蛋白NSP1负责决定宿主限制性肠道复制的表型。2)在极化上皮体外模型中表征RV细胞进入的早期事件，并确定介导细胞进入的VP4和VP7的构象。目前尚不清楚大的、无包膜的二十面体RV颗粒是如何进入极化的肠细胞的细胞质的。我们将结合使用高度特异的单抗、共聚焦显微镜和细胞生物学技术来阐明这一过程。细胞进入事件有助于不同同源和异源RV毒株的不同肠道细胞趋向性。我们假设进入过程的特征是两个轮状病毒表面蛋白的结构和细胞定位的关键变化。3)开发一种易于处理的反向遗传学系统来操纵具有复制能力的轮状病毒的基因组。为了将轮状病毒的致病机制和细胞进入研究提高到一个新的水平，需要采用方法学来直接修饰轮状病毒的双链、节段性RNA基因组。这样的系统还可能使我们和其他研究人员能够利用RV的精致肠道导向作用来设计针对其他传染病的独特靶向载体。我们的假设是，我们可以通过利用最近被验证为呼肠孤病毒和环状病毒的方法来实现这一目标。

DESCRIPTION (provided by applicant): Rotaviruses (RV) are the single most important cause of severe diarrhea in infants and young children worldwide. These viruses are also common causes of disease in healthy adults, the elderly and the immunocompromised. Virtually all mammalian species are efficiently infected with their own homologous host- species RV but not by heterologous RV. Recently, effective, live attenuated RV vaccines became commercially available, but their mechanisms of action and molecular basis for attenuation are not understood. Infection with RV is predominantly restricted to the villous epithelium of the small intestine. Of note, the basis for host-range restriction of heterologous RV replication in intestinal cells remains unknown. Due to their relatively small genomic size, host-range and tissue tropism, rotaviruses are a highly tractable model system for study of the interaction between host epithelium and enteric pathogens. RVs are also potential vectors for targeting the human small bowel epithelium and an ideal system for assessing vaccination strategies against enteric pathogens. It is our objective to continue studies of RV-host interactions with the goal of better understanding enteric microbial pathogenesis. The specific aims of the proposal are to: 1) Characterize the genetic basis for host-restricted intestinal cell tropism of selected homologous and heterologous RVs using viral reassortants in a murine model. It is our hypothesis that one of the RV surface proteins (VP4), which mediates cell entry and the RV IRF3 antagonist protein, NSP1, which blocks the host interferon response, are responsible for determining the phenotype of host restricted intestinal replication. 2) Characterize early events in RV cell entry in an in vitro model of polarized epithelium and determine the conformations of VP4 and VP7 that mediate cell entry. It is not clear how the large, non-enveloped icosahedral RV particle enters the cytoplasm of a polarized enterocyte. We will use a combination of highly specific monoclonal antibodies, confocal microscopy and cell biologic techniques to illuminate this process. Cell entry events contribute to the varying intestinal cell tropisms of different homologous and heterologous RV strains. We hypothesize that the entry process is characterized by critical changes in the structure and cellular localization of the two RV surface proteins. 3) Develop a tractable reverse-genetics system to manipulate the genome of a replication competent rotavirus. In order to take RV pathogenesis and cell entry studies to the next level, methodologies to directly modify the RV double-stranded, segmented RNA genome are needed. Such a system will also potentially enable us and other investigators to utilize the exquisite entero-tropism of RVs to design unique targeting vectors for other infectious diseases. It is our hypothesis that we can accomplish this aim by utilizing approaches recently validated for reovirus and orbiviruses.