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

项目总结/摘要 轮状病毒（RV）是非常重要的高传染性病毒，因为它们是最常见的 引起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)这将扩大我们对调节RV先天免疫逃避的分子机制的理解。 1.确定NSP 1介导的β-TrCP降解的结构基础和体内活性。 尽管RV NSP 1蛋白诱导IRF 3和/或β-TrCP降解的能力得到了充分的证明， 调节这种降解的机制是未知的。人RV NSP 1特异性靶向β-TrCP。我们 最近发现了宿主Cullin-E3连接酶复合物在NSP 1降解中的意想不到的作用， 功能协调发展的在这个目标中，我们将剖析NSP 1是如何能够劫持宿主Cullin-E3连接酶复合物，诱导β- TrCP降解，阻断NF-κB活化，从而促进同源RV复制。 2.确定菌株和宿主中VP 3降解MAVS的分子机制， 在体外和体内都有特异性表达。 我们先前表明RV感染的ssRNA副产物是胞质传感器的有效激活剂 RIG-I和MDA 5，两者都会聚于线粒体抗病毒信号蛋白（MAVS），以传递先天性 信号传导并诱导IFN表达。意想不到的初步研究结果表明，MAVS是针对 RV VP 3蛋白以宿主范围限制（HRR）方式进行蛋白酶体降解。这里我们将 在机制水平上探索来自不同RV物种的VP 3和MAVS之间的复杂相互作用， 评估VP 3介导的MAVS降解在体外和体内促进RV复制的重要性。 3.确定RV NSP 1介导的抑制STAT 1活化和肠细胞的机制 IFN对RV感染反应的起源。 尽管能够有效抑制肠上皮细胞（IEC）中I型IFN的诱导， 同源RV感染仍然在肠道中诱导大量水平的I型和III型IFN。这种干扰素的产生 表明RV必须能够破坏IFN介导的抗病毒扩增以及阻断IFN 诱导RV NSP 1有效抑制IFN介导的STAT 1磷酸化。新发现表明RV 通过消耗多种IFN受体阻断STAT 1活化，可能是通过NSP 1定向降解。RV可以 在体外也阻断未感染细胞中IFN-导向的STAT 1活化。这种效应是否也发生在体内， 未知我们建议确定RV引起的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IFN受体降解的机制决定因素， STAT 1激活的抑制，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.