Anti-interferon Activity of the Arenavirus Nucleoprotein

沙粒病毒核蛋白的抗干扰素活性

• 批准号: 8259837
• 负责人: Luis Martinez-Sobrido
• 金额: $ 44.03万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259837
• 关键词: Acute; Adverse effects; Affect; Affinity Chromatography; Amino Acids; Antiviral Agents; Arenavirus; Biochemical Genetics; Biological; Biological Assay; Biological Models; Bioterrorism; C-terminal; Calculi; Cells; Cultured Cells; Cytoplasm; Development; Disease; Exhibits; Failure; Gene Expression; Genes; Genetic Determinism; Goals; Growth; Health; Human; Immune; Immune Response Genes; Immune response; Infection; Inflammatory; Interferon Activation; Interferon Type I; Interferons; Investigation; Knowledge; Licensing; Lymphocytic choriomeningitis virus; Mass Spectrum Analysis; Mediating; Methods; Molecular; Mus; Mutate; Mutation; Natural Immunity; Nuclear; Nuclear Translocation; Nucleoproteins; Outcome; Pathogenesis; Pathway interactions; Pattern; Pattern recognition receptor; Phosphotransferases; Play; Procedures; Production; Property; Proteins; Proteomics; RNA chemical synthesis; RNA replication; Recombinants; Ribavirin; Role; Screening procedure; System; TBK1 gene; Testing; Transcriptional Activation; Vaccines; Viral; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Replication; Work; base; cell type; clinically significant; combat; helicase; human IRF3 protein; induced pluripotent stem cell; interest; interferon regulatory factor-3; mutant; neglect; particle; pathogen; prevent; promoter; protein complex; response; transcription factor; viral RNA; virus pathogenesis

DESCRIPTION (provided by applicant): The prototypic arenavirus LCMV has proven to be a Rosetta stone for the investigation of the anti-viral host immune responses, and mechanisms of virus immune evasion and persistence. In addition, evidence indicates that LCMV, worldwide distributed, is a neglected human pathogen of clinical significance. Moreover, several other arenaviruses cause hemorrhagic fever (HF) disease in humans. The failure of the host innate immune response to control virus multiplication contributes to arenaviral-induced disease. We have shown that the nucleoprotein (NP) of LCMV and HF arenaviruses inhibits activation of the transcriptional factor IRF3 and production of Type I interferon (IFN-I), a key player in innate immunity. Our long-term goal is to understand the molecular bases and biological consequences of the IFN counteracting activity associated with arenavirus NP. To this end we propose the following specific aims: Aim 1. Define the genetic determinants of LCMV-NP responsible for its IFN antagonistic function. We have identified two C-terminal domains of LCMV-NP that are critical for the anti-IFN-I activity of NP. We hypothesize that specific residues within these two regions play a critical role on the IFN antagonistic function of NP, and that this function can be segregated from the role of NP in virus replication and production of infectious progeny. We propose to conduct a comprehensive mutation- function analysis of these regions to identify single amino acid mutations in NP that abrogate its anti-IFN function without affecting other roles of NP required for arenavirus multiplication. Aim 2. Define the mechanisms by which NP exerts its IFN antagonistic activity. We have shown that LCMV-NP counteracts the IFN response by inhibiting activation and nuclear translocation of IRF3. We hypothesize that LCMV-NP achieves this by interacting with host cell proteins involved in activation of IRF3. We will pursue proteomic approaches including affinity purification methods combined with mass spectrometry procedures to identify NP-interacting cellular proteins. We will use biochemical and genetic approaches, together with functional assays, to assess the contribution of identified NP-interacting cellular proteins to the IFN antagonistic activity of NP. Aim 3. Assess the contribution of the IFN antagonistic activity of NP to virus multiplication and pathogenesis during LCMV infection of its natural host, the mouse. We will generate recombinant LCM viruses (rLCMV/NP*) containing mutated forms of NP that retain wt activity in virus RNA synthesis and infectious particle formation but lost the ability to inhibit IRF3 activation and IFN production. We hypothesize that rLCMV/NP*-infected cells will exhibit higher levels of IRF3-inducible gene expression, including IFN production, compared to LCMV/WT-infected cells. We predict this to result in altered growth properties of rLCMV/NP* with consequences for the outcome of LCMV-host interaction during acute and persistent infection. PUBLIC HEALTH RELEVANCE Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and the worldwide-distributed prototypic arenavirus LCMV is being considered as a neglected human pathogen of clinical significance. Moreover, weaponized forms of arenaviruses pose a serious threat as agents of bioterrorism. No licensed anti-arenavirus vaccines are available, and current anti-arenavirus therapy is limited to the use of ribavirin, which is only partially effective and often associated with severe side effects. Evidence indicates that a failure of the host innate immune response to control virus multiplication plays a critical role in arenaviral- induced disease. We have shown that the nucleoprotein (NP) of LCMV and HF arenaviruses inhibits activation of the transcriptional factor IRF3 and production of Type I interferon (IFN), a key player in innate immunity. Our long-term goal is to understand the molecular bases and biological consequences of the IFN counteracting activity associated with arenavirus NP. This knowledge should fuel the development of better antiviral strategies to combat human pathogenic arenaviruses.

描述（由申请方提供）：原型沙粒病毒LCMV已被证明是研究抗病毒宿主免疫应答以及病毒免疫逃避和持久性机制的Rosetta stone。此外，有证据表明，LCMV，世界各地分布，是一个被忽视的人类病原体的临床意义。此外，几种其他沙粒病毒在人类中引起出血热（HF）疾病。宿主先天免疫应答控制病毒增殖的失败促成了沙粒病毒诱导的疾病。我们已经表明，LCMV和HF沙粒病毒的核蛋白（NP）抑制转录因子IRF 3的激活和I型干扰素（IFN-I）的产生，这是先天免疫的关键因素。我们的长期目标是了解与沙粒病毒NP相关的IFN抵消活性的分子基础和生物学后果。为此，我们提出以下具体目标：目标1。确定LCMV-NP的遗传决定因素，负责其IFN拮抗功能。我们已经鉴定了LCMV-NP的两个C-末端结构域，这两个结构域对于NP的抗IFN-I活性至关重要。我们推测，这两个区域内的特定残基发挥关键作用的NP的IFN拮抗功能，这种功能可以从NP在病毒复制和生产的感染性后代的作用分离。我们建议对这些区域进行全面的突变功能分析，以确定NP中的单个氨基酸突变，这些突变废除了其抗IFN功能，而不影响沙粒病毒增殖所需的NP的其他作用。目标2.确定NP发挥其IFN拮抗活性的机制。我们已经表明，LCMV-NP通过抑制IRF 3的活化和核转位来抵消IFN应答。我们假设LCMV-NP通过与参与IRF 3激活的宿主细胞蛋白相互作用来实现这一点。我们将追求蛋白质组学的方法，包括亲和纯化方法结合质谱程序，以确定NP相互作用的细胞蛋白。我们将使用生物化学和遗传学的方法，连同功能测定，以评估所确定的NP相互作用的细胞蛋白质的NP的IFN拮抗活性的贡献。目标3.评估NP的IFN拮抗活性在LCMV感染其天然宿主小鼠期间对病毒增殖和致病性的贡献。我们将产生含有突变形式的NP的重组LCM病毒（rLCMV/NP*），所述突变形式的NP在病毒RNA合成和感染性颗粒形成中保留wt活性，但失去抑制IRF 3活化和IFN产生的能力。我们假设，rLCMV/NP* 感染的细胞将表现出更高水平的IRF 3诱导的基因表达，包括IFN的生产，相比LCMV/WT感染的细胞。我们预测这会导致rLCMV/NP* 的生长特性改变，并对急性和持续感染期间LCMV-宿主相互作用的结果产生影响。几种沙粒病毒在人类中引起出血热（HF）疾病，并且世界范围分布的原型沙粒病毒LCMV被认为是具有临床意义的被忽视的人类病原体。此外，沙粒病毒的武器化形式作为生物恐怖主义制剂构成严重威胁。没有获得许可的抗沙粒病毒疫苗可用，目前的抗沙粒病毒治疗仅限于使用利巴韦林，其仅部分有效且通常与严重副作用相关。有证据表明，宿主先天免疫应答控制病毒增殖的失败在沙粒病毒诱导的疾病中起关键作用。我们已经表明，LCMV和HF沙粒病毒的核蛋白（NP）抑制转录因子IRF 3的激活和I型干扰素（IFN）的产生，I型干扰素是先天免疫的关键因素。我们的长期目标是了解与沙粒病毒NP相关的IFN抵消活性的分子基础和生物学后果。这一知识应该推动更好的抗病毒策略的发展，以打击人类致病性沙粒病毒。

项目成果

Transcription and replication mechanisms of Bunyaviridae and Arenaviridae L proteins.

• DOI: 10.1016/j.virusres.2017.01.018
• 发表时间: 2017-04-15
• 期刊: Virus research
• 影响因子: 5
• 作者: Ferron F;Weber F;de la Torre JC;Reguera J
• 通讯作者: Reguera J

Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays.

• DOI: 10.3390/v10110655
• 发表时间: 2018-11-20
• 期刊: Viruses
• 作者: Caì Y;Iwasaki M;Beitzel BF;Yú S;Postnikova EN;Cubitt B;DeWald LE;Radoshitzky SR;Bollinger L;Jahrling PB;Palacios GF;de la Torre JC;Kuhn JH
• 通讯作者: Kuhn JH

Generation of Bi-Reporter-Expressing Tri-Segmented Arenavirus.

• DOI: 10.1007/978-1-0716-2453-1_17
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Ye, Chengjin;Martinez-Sobrido, Luis
• 通讯作者: Martinez-Sobrido, Luis

Inhibition of multiplication of the prototypic arenavirus LCMV by valproic acid.

丙戊酸抑制原型沙粒病毒 LCMV 的增殖。

• DOI: 10.1016/j.antiviral.2013.05.012
• 发表时间: 2013
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Vázquez-Calvo,Ángela;Martín-Acebes,MiguelA;Sáiz,Juan-Carlos;Ngo,Nhi;Sobrino,Francisco;delaTorre,JuanCarlos
• 通讯作者: delaTorre,JuanCarlos

Reverse genetics approaches for the development of mammarenavirus live-attenuated vaccines.

• DOI: 10.1016/j.coviro.2020.06.011
• 发表时间: 2020-10
• 期刊: Current opinion in virology
• 影响因子: 5.9
• 作者: Ye C;de la Torre JC;Martinez-Sobrido L
• 通讯作者: Martinez-Sobrido L