Novel Hantavirus Virulence Determinants

 描述（由申请方提供）：汉坦病毒（HV）感染毛细血管内皮细胞（EC）衬里，并以非溶解性方式引起血管渗漏。安第斯山脉病毒（ANDV）和Sin Nombre病毒（SNV）感染几乎所有的肺EC，导致35%致命的急性肺水肿（HV肺综合征-HPS）。然而，ANDV是唯一一种人与人传播的HV，也是唯一一种在叙利亚仓鼠中引起致命的HPS样疾病的HV。我们发现ANDV核衣壳（N）蛋白独特地调节干扰素（IFN）诱导，表明ANDV编码一种新的毒力决定簇。在感染前或感染后早期加入的IFN可抑制HV，并且为了在人EC中复制，致病性HV调节早期IFN诱导。除PHV外，HV Gn蛋白含有抑制RIG-I/TBK 1介导的IRF 3磷酸化和IFNβ诱导的胞质尾区（GnT）元件。相比之下，来自SNV、NY-1V、HTNV和PHV的N蛋白不能抑制RIG-I/TBK 1指导的IFN诱导。出乎意料的是，ANDV N蛋白抑制RIGI/TBK 1指导的IFN诱导。而MAPV的N蛋白不参与IFN信号的调节。美国ANDV同源物与人类疾病无关。因此，只有ANDV表达调节RIGI/MDA 5/MAVS和TBK 1 IFN信号传导应答的N蛋白。进一步的分析确定ANDV N抑制TBK 1自身磷酸化，而Gn阻止活化的pTBK 1磷酸化IRF 3。因此，ANDV独特地表达IFN调节N蛋白，其可与Gn协同作用以调节连续的TBK 1信号传导步骤。与此一致，N和GnGc蛋白定位于ER/cisGolgi，其中TRAF 3/TBK 1/TANK复合物受多种因子调节。我们最近的蛋白质组学分析N与EC蛋白的相互作用定义TRIM 21作为一个假定的ANDV N结合伴侣。TRIM 21调节TBK 1-IRF 3信号转导应答，表明ANDV N可能靶向TRIM 21以抑制IFN诱导。这进一步表明Gn蛋白可以独立地或协同地靶向TRIM 21功能以抑制TBK 1-IRF 3信号传导。我们的数据定义N蛋白作为一种新的ANDV特异性毒力决定因子。这可以解释为什么ANDV在叙利亚仓鼠中独特地引起病毒血症和致命的HPS样疾病，并且是唯一在人与人之间传播的HV。我们最近的发现，ANDV反向遗传学的发展以及我们与Jay Hooper（USAMRIID）的合作关系使我们能够提出定义N和Gn突变，这些突变可以减弱ANDV并防止叙利亚仓鼠中的致命HPS。我们建议定义IFN调节HV N和Gn蛋白内的毒力决定簇以及N/Gn调节RIG-I/TBK 1信号通路的机制。我们还将确定是否在N和Gn内突变IFN调节元件或用MAPV再确认ANDV 1）减少人EC中的ANDV复制; 2）在致死性叙利亚仓鼠HPS模型中减弱ANDV指导的HPS和3）保护仓鼠免受致死性ANDV感染。这些研究有可能开发减毒ANDV作为潜在的疫苗。

 DESCRIPTION (provided by applicant): Hantaviruses (HVs) infect the endothelial cell (EC) lining of capillaries and nonlytically cause vascular leakage. Andes virus (ANDV) and Sin Nombre virus (SNV) infect virtually all pulmonary ECs resulting in acute pulmonary edema that is 35% fatal (HV pulmonary syndrome-HPS). However, ANDV is the only HV spread person to person and the only HV that causes lethal HPS-like disease in Syrian hamsters. We found that the ANDV nucleocapsid (N) protein uniquely regulates interferon (IFN) induction, suggesting that ANDV encodes a novel virulence determinant. HVs are inhibited by IFN added prior to or early after infection, and in order to replicate in human ECs pathogenic HVs regulate early IFN induction. Except for PHV, HV Gn proteins contain cytoplasmic tail (GnT) elements that inhibit RIG-I/TBK1 directed IRF3 phosphorylation and IFNβ induction. In contrast, N proteins from SNV, NY-1V, HTNV and PHV fail to inhibit RIG-I/TBK1 directed IFN induction. Unexpectedly, the ANDV N protein inhibited RIGI/TBK1 directed IFN induction. Yet, IFN signaling was not regulated by N protein from MAPV, a S. American ANDV homologue not linked to human disease. Thus only ANDV expresses an N protein that regulates RIGI/MDA5/MAVS and TBK1 IFN signaling responses. Further analysis determined that ANDV N inhibits TBK1 autophosphorylation while Gn prevents activated pTBK1 from phosphorylating IRF3. Thus ANDV uniquely expresses an IFN regulating N protein that may act in concert with Gn to regulate sequential TBK1 signaling steps. Consistent with this, N and GnGc proteins localize to the ER/cisGolgi where TRAF3/TBK1/TANK complexes are regulated by a plethora of factors. Our recent proteomics analysis of N interactions with EC proteins defined TRIM21 as a putative ANDV N binding partner. TRIM21 regulates TBK1-IRF3 signaling responses suggesting that ANDV N may target TRIM21 to inhibit IFN induction. This further suggests that Gn proteins may independently or synergistically target TRIM21 functions to inhibit TBK1-IRF3 signaling. Our data defines N protein as a novel ANDV-specific virulence determinant. This may explain why ANDV uniquely causes viremia and lethal HPS-like disease in Syrian hamsters and is the only HV that is spread person to person. Our recent findings, the development of ANDV reverse genetics and our partnership with Jay Hooper (USAMRIID) permit us to propose defining N and Gn mutations that attenuate ANDV and prevent lethal HPS in Syrian hamsters. We propose defining IFN regulating virulence determinants within HV N and Gn proteins and mechanisms by which N/Gn regulate RIG-I/TBK1 signaling pathways. We will also determine whether mutating IFN regulating elements within N and Gn or reasserting ANDV with MAPV 1) reduces ANDV replication in human ECs; 2) attenuates ANDV directed HPS in the lethal Syrian hamster HPS model and 3) protects hamsters from lethal ANDV infection. These studies are likely to develop attenuated ANDVs as potential vaccines.