Reverse Genetics of Arenaviruses.

沙粒病毒的反向遗传学。

• 批准号: 8009824
• 负责人: Juan C. de la Torre
• 金额: $ 41.79万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-15 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009824
• 关键词: Acute; Adverse effects; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Amino Acids; Antiviral Agents; Arenavirus; Arenavirus Infections; Attention; Binding; Biochemical Genetics; Biological Assay; Biological Models; Biology; Bioterrorism; Cell Culture Techniques; Cells; Cellular biology; Clinical; Collaborations; Complement; Complex; Cues; Development; Disease; E protein; Engineering; Equipment; Experimental Models; Figs - dietary; Funding; Genes; Genetic; Genetic Screening; Goals; Growth; Health; Howard Temin Award; Human; Immunology; Infection; Institutes; Investigation; Knowledge; Laboratories; Libraries; Licensing; Logistics; Lymphocytic choriomeningitis virus; Mass Spectrum Analysis; Mediating; Medical Research; Minor; Molecular; Molecular Biology; One-Step dentin bonding system; Pathogenesis; Pathway interactions; Peptides; Physiological; Principal Investigator; Procedures; Production; Proteins; Proteomics; RNA chemical synthesis; Recommendation; Research; Research Personnel; Ribavirin; Role; Screening procedure; Small Interfering RNA; Streptavidin; Study Section; System; Technology; Testing; Text; Time; Update; Vaccines; Viral; Viral Hemorrhagic Fevers; Virulence; Virus Diseases; base; clinically significant; combat; editorial; experience; follow-up; genome-wide; high throughput screening; interest; meetings; mouse model; neglect; novel; particle; pathogen; plasma protein Z; positional cloning; programs; response; viral RNA; virus host interaction

DESCRIPTION (provided by applicant): Arenaviruses merit significant interest both as tractable experimental model systems to study acute and persistent viral infections and as clinically important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans, whereas the prototypic Arenavirus LCMV is a superb workhorse for the investigation of basic concepts in the fields of viral immunology and pathogenesis. In addition, evidence indicates that LCMV is a neglected human pathogen of clinical significance. Our long- term objective is to obtain a detailed understanding of the Arenavirus molecular and cell biology. This knowledge will contribute to the elucidation of Arenavirus-host interactions and associated diseases, and facilitate the development of effective strategies to combat Arenavirus infections. To this end we have developed a reverse genetics system for LCMV that provides us with a novel and powerful approach for the investigation of Arenavirus biology. The focus of this proposal is to functionally characterize viral and cellular proteins, and their interactions, which mediate control of Arenavirus RNA synthesis, particle formation and viral budding. Our specific aims are: 1. To Assess the role of Z-NP In control of viral RNA synthesis. We will test the hypothesis that Z- NP interaction is responsible for the inhibitory activity of Z on viral RNA synthesis. We will use biochemical and genetic approaches to identify the regions of Z and NP required for Z-NP interaction, and use reverse genetic approaches to examine the functional consequences of disrupting Z-NP interaction. 2. Determine the role of Z-GP interaction in production of arenavirus infectious particles. We have demonstrated that production of infectious VLP requires Z and GP, and shown that Z and GP interact. We will define biochemically and functionally this Z-GP interaction. We will test the hypothesis that the correct Z-GPcx association is required for the production of infectious particles, and that this interaction is mediated by defined regions within Z and the GP-2 component of the GP complex (GPcx). 3. Determined the function of Z L domain motifs during the natural course of Arenavirus infection. We will use reverse genetics to rescue rLCMV carrying Z proteins with different types of L-domains. These rLCMV will be used in cell culture and mice models of infection to test the hypothesis that the type of L- domain motifs present in Z influences viral growth and virulence. 4. Identify and functionally characterize host proteins that influence Z-mediated budding. We will test the hypothesis that Z proteins containing different L-domains engage distinct subsets of Class E proteins of the MVB pathway, and that these differences influence virus-host interactions and associated diseases. We will use proteomic approaches to define the Z interactome. As a complementary approach we will use siRNA-based screenings to identify cellular factors that contribute to Z-mediated budding. We will use biochemical, genetics and functional assays to assess the relevance in Arenavirus biology of candidates initially identified by proteomic and genetic approaches. PUBLIC HEALTH RELEVANCE: Several arenaviruses cause hemorrhagic fever (HF) disease in humans, and mounting evidence indicates that the worldwide-distributed prototypic arenavirus LCMV is 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. Therefore it is important to develop novel antiviral strategies to combat arenaviral infections. A detailed understanding of the arenavirus molecular will facilitate this task and cell biology, which is the long-term goal of the studies proposed in this application.

描述（由申请方提供）：沙粒病毒作为研究急性和持续性病毒感染的易处理实验模型系统以及作为临床上重要的人类病原体都值得关注。几种沙粒病毒在人类中引起出血热（HF）疾病，而原型沙粒病毒LCMV是研究病毒免疫学和发病机理领域中的基本概念的极好的主力。此外，有证据表明LCMV是一种被忽视的具有临床意义的人类病原体。我们的长期目标是获得沙粒病毒分子和细胞生物学的详细了解。这些知识将有助于阐明沙粒病毒与宿主的相互作用和相关疾病，并促进制定有效的战略来对抗沙粒病毒感染。为此，我们开发了一个LCMV的反向遗传学系统，为我们提供了一个新的和强大的方法来研究沙粒病毒的生物学。该提案的重点是功能性表征病毒和细胞蛋白及其相互作用，介导沙粒病毒RNA合成，颗粒形成和病毒出芽的控制。我们的具体目标是：1.评价Z-NP在控制病毒RNA合成中的作用。我们将检验Z-NP相互作用是Z对病毒RNA合成的抑制活性的原因这一假设。我们将使用生物化学和遗传学方法来确定Z-NP相互作用所需的Z和NP区域，并使用反向遗传学方法来研究破坏Z-NP相互作用的功能后果。2.确定Z-GP相互作用在沙粒病毒感染性颗粒产生中的作用。我们已经证明，生产传染性VLP需要Z和GP，并表明Z和GP相互作用。我们将从生物化学和功能上定义这种Z-GP相互作用。我们将检验以下假设：感染性颗粒的产生需要正确的Z-GPcx结合，并且这种相互作用由Z内的定义区域和GP复合物（GPcx）的GP-2组分介导。3.确定Z-L结构域基序在沙粒病毒自然感染过程中的功能。我们将使用反向遗传学来拯救携带具有不同类型L结构域的Z蛋白的rLCMV。这些rLCMV将用于细胞培养和小鼠感染模型中，以检验Z中存在的L结构域基序的类型影响病毒生长和毒力的假设。4.鉴定和功能表征影响Z介导的出芽的宿主蛋白。我们将测试的假设，Z蛋白含有不同的L-结构域从事不同的子集的E类蛋白的MVB途径，这些差异影响病毒-宿主的相互作用和相关疾病。我们将使用蛋白质组学方法来定义Z相互作用组。作为一种补充方法，我们将使用基于siRNA的筛选来鉴定有助于Z介导的出芽的细胞因子。我们将使用生物化学，遗传学和功能测定来评估最初通过蛋白质组学和遗传学方法鉴定的候选物在沙粒病毒生物学中的相关性。公共卫生关系： 几种沙粒病毒引起人类出血热（HF）疾病，越来越多的证据表明，世界范围内分布的原型沙粒病毒LCMV是一种被忽视的人类病原体的临床意义。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。因此，重要的是开发新的抗病毒策略来对抗沙粒病毒感染。对沙粒病毒分子的详细了解将有助于这项任务和细胞生物学，这是本申请中提出的研究的长期目标。

项目成果

Mapping the landscape of the lymphocytic choriomeningitis virus stable signal peptide reveals novel functional domains.

绘制淋巴细胞脉络丛脑膜炎病毒稳定信号肽的图谱揭示了新的功能域。

• DOI: 10.1128/jvi.02759-06
• 发表时间: 2007
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Saunders,AprilA;Ting,JoeyPC;Meisner,Jeffrey;Neuman,BenjaminW;Perez,Mar;delaTorre,JuanCarlos;Buchmeier,MichaelJ
• 通讯作者: Buchmeier,MichaelJ

Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.

新型沙粒病毒增殖小分子抑制剂的鉴定和作用机制。

• DOI: 10.1128/jvi.01587-15
• 发表时间: 2015
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Ngo,Nhi;Henthorn,KristinaSchimmelpfeng;Cisneros,MariaIsabel;Cubitt,Beatrice;Iwasaki,Masaharu;delaTorre,JuanC;Lama,Juan
• 通讯作者: Lama,Juan

Development of Recombinant Arenavirus-Based Vaccines.

基于重组沙粒病毒的疫苗的开发。

• DOI: 10.1007/978-1-4939-6869-5_8
• 发表时间: 2017
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Martínez-Sobrido,Luis;delaTorre,JuanCarlos
• 通讯作者: delaTorre,JuanCarlos

Novel strategies for development of hemorrhagic fever arenavirus live-attenuated vaccines.

开发出血热沙粒病毒减毒活疫苗的新策略。

• DOI: 10.1080/14760584.2016.1182024
• 发表时间: 2016
• 期刊: Expert review of vaccines
• 影响因子: 6.2
• 作者: Martinez-Sobrido,Luis;delaTorre,JuanCarlos
• 通讯作者: delaTorre,JuanCarlos

Arenavirus budding.

• DOI: 10.1155/2011/180326
• 发表时间: 2011
• 期刊: Advances in virology
• 影响因子: 2.2
• 作者: Urata S;de la Torre JC
• 通讯作者: de la Torre JC