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（由申请人提供）：沙粒病毒LCMV原型已被证明是研究抗病毒宿主免疫反应以及病毒免疫逃避和持续机制的罗塞塔石。此外，有证据表明，全球分布的LCMV是一种被忽视的具有临床意义的人类病原体。此外，其他几种沙粒病毒引起人类出血热（HF）疾病。宿主先天免疫反应控制病毒增殖的失败有助于沙粒病毒诱导的疾病。我们已经证明LCMV和HF沙粒病毒的核蛋白（NP）抑制转录因子IRF3的激活和I型干扰素（IFN-I）的产生，IFN-I是先天免疫的关键角色。我们的长期目标是了解与沙粒病毒NP相关的干扰素拮抗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。我们已经确定了LCMV-NP的两个c端结构域，它们对NP的抗ifn - i活性至关重要。我们假设这两个区域内的特定残基对NP的IFN拮抗功能起关键作用，并且该功能可以与NP在病毒复制和感染性子代产生中的作用分离开来。我们建议对这些区域进行全面的突变功能分析，以确定NP中单个氨基酸突变，这些突变可以取消其抗ifn功能，但不会影响NP在沙粒病毒增殖所需的其他作用。目标2。定义NP发挥其IFN拮抗活性的机制。我们已经证明LCMV-NP通过抑制IRF3的激活和核易位来抵消IFN的反应。我们假设LCMV-NP通过与参与IRF3激活的宿主细胞蛋白相互作用来实现这一目标。我们将采用蛋白质组学方法，包括亲和纯化方法结合质谱法来鉴定np相互作用的细胞蛋白。我们将使用生化和遗传方法，以及功能分析，来评估鉴定的NP相互作用细胞蛋白对NP的IFN拮抗活性的贡献。目标3。评估NP对LCMV感染自然宿主小鼠时病毒增殖和发病机制的IFN拮抗活性的贡献。我们将生成含有NP突变形式的重组LCM病毒（rLCMV/NP*），这些NP在病毒RNA合成和感染性颗粒形成中保留wt活性，但失去了抑制IRF3激活和IFN产生的能力。我们假设，与LCMV/ wt感染的细胞相比，rLCMV/NP*感染的细胞将表现出更高水平的irf3诱导基因表达，包括IFN的产生。我们预测这将导致rLCMV/NP*生长特性的改变，从而影响急性和持续性感染期间lcmv -宿主相互作用的结果。几种沙粒病毒引起人类出血热（HF）疾病，而世界范围内分布的原型沙粒病毒LCMV被认为是一种被忽视的具有临床意义的人类病原体。此外，武器化的沙粒病毒作为生物恐怖主义的媒介构成严重威胁。目前还没有获得许可的抗沙粒病毒疫苗，目前的抗沙粒病毒治疗仅限于使用利巴韦林，这种药物仅部分有效，而且往往伴有严重的副作用。有证据表明，宿主先天免疫反应控制病毒增殖的失败在沙粒病毒诱导的疾病中起着关键作用。我们已经证明LCMV和HF沙粒病毒的核蛋白（NP）抑制转录因子IRF3的激活和I型干扰素（IFN）的产生，IFN是先天免疫的关键角色。我们的长期目标是了解与沙粒病毒NP相关的干扰素拮抗活性的分子基础和生物学后果。这一知识应有助于开发更好的抗病毒策略来对抗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