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Defining ANDV Virulence and Attenuation Mechanisms

定义 ANDV 毒力和减毒机制

基本信息

批准号：
10054155
负责人：
Erich R Mackow
金额：
$ 64.19万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-11-25 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10054155
关键词：
Acute American Andes Virus Attenuated Binding Blood Vessels Blood capillaries Complex Cytoplasmic Tail Data Disease Disease model Elements Endothelial Cells Extravasation Family member Genes Hamsters Hantavirus Hantavirus Infections Hantavirus Pulmonary Syndrome Human IRF3 gene Immune response Infection Innate Immune Response Interferon Type I Interferon-alpha Interferons Kinetics Lung Mesocricetus auratus Mutate Mutation Nonlytic Pathogenicity Pathway interactions Persons Phosphorylation Plasmids Proteins Proteomics Pulmonary Edema RNA Viruses Reassortant Viruses Regulation Renilla Reporter Reporting Role Signal Pathway Signal Transduction Sin Nombre virus TBK1 gene TNF receptor-associated factor 3 TRIM Family Testing Viremia Virulence Virus Diseases attenuation human pathogen mutant novel prevent response reverse genetics sensor synergism transcription factor vaccine candidate vaccine development vaccine evaluation virtual

项目摘要

Hantaviruses (HVs) predominantly 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(109) and cause 35% fatal acute pulmonary edema (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 hamsters. Pathogenic HVs regulate early innate immune responses in order to successfully replicate in ECs and we recently revealed that ANDV contains an additional interferon (IFN) regulating determinant that explains unique ANDV spread and virulence and is a potential target for attenuating ANDV. 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. Nonpathogenic PHV, fails to regulate IFN induction or replicate in human ECs, and failed IFN regulation explains why PHV is not a human pathogen. 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 TULV, SNV, NY-1V, HTNV and PHV fail to inhibit RIG-I/TBK1 directed IFN induction. Unpredictably, the ANDV N protein inhibited RIGI/TBK1 directed IFN induction. In contrast, N protein from a nonpathogenic S. American HV, MAPV, failed to inhibit IFN signaling, yet MAPV N differs by only 12 dissimilar residues from ANDV N. ANDV N protein inhibits TBK1 autophosphorylation that is required for TBK1 phosphorylation of IRF3 and our recent proteomics screen found that ANDV N binds a single IFN regulating protein in ECs, TRIM21. Reciprocal TRIM21 IPs validated ANDV N, but not TULV N, as a TRIM21 binding partner. Preliminary data indicates that mutating ANDV residues to MAPV N residues prevents IFN regulation and TRIM21 binding, and provides a mechanism for attenuating ANDV. Our findings suggest that IFN regulation by the ANDV N protein is a novel virulence determinant. This may explain why ANDV uniquely causes viremia and lethal HPS disease in Syrian hamsters and why ANDV is the only HV spread from person to person. These findings permit us to propose defining the role of N and Gn directed IFN regulation in attenuating ANDV and preventing lethal HPS in Syrian hamsters. We define mechanisms by which N and Gn proteins inhibit RIG-I/TBK1 signaling pathways and identify mutations that abolish regulation. We evaluate ANDV reassortants and mutants for attenuation in a lethal Syrian hamster HPS disease model. We define HV virulence determinants by identifying N/Gn residues of ANDV, MAPV & SNV required to regulate RIG-I/TBK1/IRF3 signaling pathways. We analyze ANDV regulation of IFN inductionin ECs, ANDV N regulation of TRIM21, and N/Gn synergy in IFN regulation. ANDV/MAPV reassortants and N/Gn-ΔIFN rANDV mutants are evaluated for their attenuation and ability to protect Syrian hamsters from Wt ANDV infection.

汉坦病毒 (HV) 主要感染毛细血管的内皮细胞 (EC) 内层，并以非溶解方式引起血管渗漏。安第斯病毒 (ANDV) 和 Sin Nombre 病毒 (SNV) 几乎感染所有肺内皮细胞 (109) 和导致 35% 致命的急性肺水肿（HV 肺综合征 -HPS）。然而，ANDV 是唯一的 HV 在人与人之间传播，也是唯一一种在仓鼠中引起致命性 HPS 样疾病的高压病毒。致病性高压病毒调节早期先天免疫反应，以便在 EC 中成功复制，我们最近发现 ANDV 含有额外的干扰素 (IFN) 调节决定因子，可解释 ANDV 的独特传播和毒力强，是减毒 ANDV 的潜在目标。 HV 被感染前或感染后早期添加的 IFN 抑制，以便在人类 EC 中复制致病性 HV 调节早期 IFN 诱导。非致病性 PHV，无法调节 IFN 诱导或复制人类 EC 和失败的 IFN 调节解释了为什么 PHV 不是人类病原体。 PHV、HV Gn 除外蛋白质含有抑制 RIG-I/TBK1 指导的 IRF3 磷酸化的细胞质尾 (GnT) 元件， IFNβ诱导。相反，来自 TULV、SNV、NY-1V、HTNV 和 PHV 的 N 蛋白无法抑制 RIG-I/TBK1 定向干扰素诱导。出乎意料的是，ANDV N 蛋白抑制了 RIGI/TBK1 指导的 IFN 诱导。在相比之下，来自非致病性南美洲HV（MAPV）的N蛋白未能抑制IFN信号传导，但MAPV N 与 ANDV N 仅有 12 个不同残基不同。 ANDV N 蛋白抑制 TBK1 自磷酸化，这是 IRF3 和 TBK1 磷酸化所必需的我们最近的蛋白质组学筛选发现 ANDV N 与 EC 中的单个 IFN 调节蛋白 TRIM21 结合。相互的 TRIM21 IP 验证了 ANDV N 作为 TRIM21 结合伴侣，但没有验证 TULV N。初步数据表明将 ANDV 残基突变为 MAPV N 残基会阻止 IFN 调节和 TRIM21 结合，并且提供了一种减弱 ANDV 的机制。我们的研究结果表明，ANDV N 蛋白对 IFN 的调节是一种新的毒力决定因素。这可以解释为什么 ANDV 独特地导致病毒血症和致命的 HPS 疾病以及为什么 ANDV 是唯一在人与人之间传播的 HV。这些发现使我们能够建议定义 N 和 Gn 导向的 IFN 调节在减弱 ANDV 和预防致命 HPS 中的作用在叙利亚仓鼠中。我们定义了 N 和 Gn 蛋白抑制 RIG-I/TBK1 信号通路的机制并识别废除监管的突变。我们评估 ANDV 重配体和突变体的减毒作用致命的叙利亚仓鼠 HPS 疾病模型。我们通过识别 ANDV、MAPV 和 SNV 所需的 N/Gn 残基来定义 HV 毒力决定因素调节 RIG-I/TBK1/IRF3 信号通路。我们分析了 ANDV 对 EC 中 IFN 诱导的调节，ANDV N TRIM21 的调节，以及 N/Gn 在 IFN 调节中的协同作用。 ANDV/MAPV 重配体和 N/Gn-ΔIFN rANDV 评估突变体的减毒能力和保护叙利亚仓鼠免受 Wt ANDV 感染的能力。