Regulation of Rotavirus Replication, Virulence, and Host Range Restriction by the Innate Immune System

项目摘要/摘要 轮状病毒(RV)是高度传染性的病毒，因为它们是最常见的 幼儿严重胃肠炎的原因。我们将讨论轮状病毒学的三个基本主题 (RV)这将扩大我们对RV先天性免疫逃避的分子机制的理解。 1.确定NSP1介导的β-TrCP降解的结构基础和体内活性。 尽管RVNSP1蛋白有能力诱导IRF3和/或β-TrCP降解，但 调节这种降解的机制尚不清楚。人RV NSP1特异性靶向β-TrCP。我们 我们最近发现了宿主cullin-E3连接酶复合体在nsp1的S降解中的一个意想不到的作用 功能。在这个目标中，我们将剖析NSP1如何能够劫持宿主cullin-E3连接酶复合体，诱导β-E3- TrCP降解，阻断NF-κB激活，从而促进同源轮状病毒复制。 2.确定VP3在菌株和宿主中降解MAV的分子机制 在体外和体内都有独特的时尚。 我们先前已经证明rv感染的副产物ssrna是胞浆感受器的有效激活剂。 RIG-I和MDA5，两者都汇聚在线粒体抗病毒信号蛋白(MAV)上，传递先天的 信号转导和诱导干扰素表达。出人意料的初步发现表明，小牛的目标是 RV VP3蛋白以宿主范围受限(HRR)的方式降解蛋白酶体。在这里，我们将 在机制水平上探索VP3和不同RV物种的MAV之间的复杂相互作用 评价VP3介导的MAVs降解在促进RV体外和体内复制中的重要性。 3.确定RV NSP1介导的抑制STAT1活性和肠道细胞的机制 轮状病毒感染后干扰素应答的来源。 尽管有能力有效地抑制肠上皮细胞(IECS)中I型干扰素的诱导， 同源轮状病毒感染仍然在肠道中诱导大量的I型和III型IFN。这种干扰素的产生 提示轮状病毒必须能够破坏干扰素介导的抗病毒扩增，并阻断干扰素 归纳法。RV NSP1能有效抑制干扰素介导的STAT1磷酸化。新发现表明RV 通过耗尽多个干扰素受体来阻断STAT1的激活，可能是通过NSP1引导的降解。房车可以 在体外也可以阻断干扰素诱导的未感染细胞中STAT1的激活。这种效应是否也会在体内发生，目前尚不清楚 未知。我们建议鉴定RV诱导的I型和III型IFN的造血细胞和IEC来源 感染。我们还将研究RV介导的干扰素受体降解的机制决定因素和 抑制STAT1的激活，并确定这些功能的差异是否有助于RV HRR。

PROJECT SUMMARY/ABSTRACT Rotaviruses (RVs) are highly infectious viruses of great importance because they are the most common cause of severe gastroenteritis in young children. We will address three fundamental topics in rotavirology (RV) that will expand our understanding of the molecular mechanisms regulating RV innate immune evasion. 1. Determine the structural basis and in vivo activity of NSP1-mediated β-TrCP degradation. Despite the RV NSP1 protein's well-documented ability to induce IRF3 and/or β-TrCP degradation, the mechanisms regulating this degradation are unknown. Human RV NSP1s specifically target β-TrCP. We have recently identified an unexpected role of the host Cullin-E3 ligase complex in NSP1's degradative functions. In this aim we will dissect how NSP1 is able to hijack the host Cullin-E3 ligase complex, induce β- TrCP degradation, block NF-κB activation and thereby promote homologous RV replication. 2. Identify the molecular mechanisms underlying MAVS degradation by VP3 in a strain- and host- specific fashion both in vitro and in vivo. We previously showed that ssRNA byproducts from RV infection are potent activators of cytosolic sensors RIG-I and MDA5, both of which converge on mitochondrial antiviral signaling protein (MAVS) to relay innate signaling and induce IFN expression. Unexpected preliminary findings suggest that MAVS is targeted for proteasomal degradation by the RV VP3 protein in a host range restricted (HRR) manner. Here we will explore the complex interplay between VP3 and MAVS from different RV species at a mechanistic level and evaluate the importance of VP3-mediated MAVS degradation in promoting RV replication in vitro and in vivo. 3. Identify the mechanism of RV NSP1-mediated inhibition of STAT1 activation and the intestinal cell origin of the IFN responses to RV infection. Despite the ability to efficiently suppress the induction of type I IFN in intestinal epithelial cells (IECs), homologous RV infection still induces substantial levels of type I and III IFNs in the gut. This IFN production suggests that RVs must be able to subvert IFN-mediated antiviral amplification as well as blocking IFN induction. RV NSP1 efficiently inhibits IFN-mediated STAT1 phosphorylation. New findings indicate RV blocks STAT1 activation by depleting multiple IFN receptors, likely by NSP1-directed degradation. RVs can also block IFN-directed STAT1 activation in uninfected cells in vitro. Whether this effect also occurs in vivo is unknown. We propose to identify the hematopoietic cell and IEC origins of type I and III IFNs elicited by RV infection. We will also examine the mechanistic determinants of RV-mediated IFN receptor degradation and inhibition of STAT1 activation and determine if differences in these functions contribute to RV HRR.