Novel Hantavirus Virulence Determinants

 描述(申请人提供)：汉坦病毒(HV)感染毛细血管内皮细胞(EC)衬里，非溶血性导致血管渗漏。安第斯病毒(Andes Virus，ANDV)和新诺布雷病毒(Sin Norbre Virus，SNV)感染几乎所有的肺内皮细胞，导致急性肺水肿，致死率为35%(HV肺综合征-HPS)。然而，ANDV是唯一在人与人之间传播的HV，也是唯一在叙利亚仓鼠中导致致命性HPS样疾病的HV。我们发现ANDV核衣壳(N)蛋白唯一地调节干扰素的诱导，这表明ANDV编码一种新的毒力决定因素。在感染之前或感染后早期，加入干扰素可以抑制HV，并且为了在人内皮细胞中复制，致病HV调节早期干扰素的诱导。除猪瘟病毒外，HV Gn蛋白还含有胞质尾部元件，可抑制Rig-I/Tbk1诱导的irf3磷酸化和干扰素β的诱导。相反，SNV、NY-1V、HTNV和PHV的N蛋白不能抑制RIG-I/TBK1诱导的干扰素诱导。出乎意料的是，ANDV N蛋白抑制了RIGI/TBK1诱导的干扰素。然而，干扰素信号不受来自MAPV的N蛋白的调节，MAPV是一种与人类疾病无关的南美ANDV同源物。因此，只有ANDV表达调节RIGI/MDA5/MAVS和TBK1干扰素信号反应的N蛋白。进一步的分析表明，ANDV N抑制了TBK1的自动磷酸化，而Gn阻止了活化的pTBK1对IRF3的磷酸化。因此，ANDV独特地表达了一种干扰素调节N蛋白，该蛋白可能与Gn协同作用来调节顺序的TBK1信号步骤。与此一致，N和GnGc蛋白定位于ER/cisGolgi，其中TRAF3/TBK1/TANK复合体受多种因素调节。我们最近对N与EC蛋白相互作用的蛋白质组学分析将TRIM21定义为ANDV N结合伙伴。TRIM21调节TBK1-IRF3信号反应，提示ANDV N可能针对TRIM21抑制干扰素的诱导。这进一步表明，Gn蛋白可能独立地或协同地靶向TRIM21功能，以抑制TBK1-IRF3信号转导。我们的数据将N蛋白定义为一种新的ANDV特异性毒力决定因素。这可能解释了为什么ANDV独特地导致叙利亚仓鼠的病毒血症和致命性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)的合作使我们能够提出定义N和Gn突变来减弱ANDV并防止叙利亚仓鼠的致死性HPS。我们建议定义干扰素调节HV-N和Gn蛋白中的毒力决定因素，以及N/Gn调节RIG-I/TBK1信号通路的机制。我们还将确定在N和Gn内突变干扰素调节元件或将ANDV与MAPV一起重新断言1)减少ANDV在人内皮细胞中的复制；2)在致死的叙利亚仓鼠HPS模型中减弱ANDV导向的HPS，以及3)保护仓鼠免受ANDV的致死性感染。这些研究很可能开发出减毒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.