Enterovirus RNA translation and replication.

肠道病毒RNA翻译和复制。

基本信息

批准号：
8887567
负责人：
DAVID J BARTON
金额：
$ 38.24万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8887567
关键词：
Affect Alanine Amino Acid Sequence Anti-Bacterial Agents Antiviral Agents Apoptosis Aseptic Meningitis Bioinformatics Biological Biological Assay Cell-Free System Cells Child Cleaved cell Clinic Codon Nucleotides Collaborations Communities Complementary DNA Conjunctivitis Conserved Sequence Coxsackie Viruses Cytoplasm DNA-Directed RNA Polymerase Data Disease Double-Stranded RNA Drug Targeting Echovirus 6 Encephalitis Endoribonucleases Enterovirus Enzymes Exanthema Family Picornaviridae Fever Frequencies Funding Genetic Recombination Genetic Transcription Genetic Translation Genomic approach Genomics Hand, Foot and Mouth Disease Human Human poliovirus Immune response Individual Induced Mutation Ions Location Longevity Malignant Neoplasms Measures Mediating Messenger RNA Metals Methodology Methods Molecular Molecular Target Mutation Myocarditis Nature Outcome Paralysed Pathogenesis Pathway interactions Phenotype Phosphodiesterase I Pneumonia Poliovirus Vaccines Polioviruses Polyadenylation Polymerase Process RNA RNA Sequences RNA Stability RNA Viruses RNA chemical synthesis RNA replication Recombinants Research Resources Rhinovirus Ribavirin Ribonucleases Ribosomal RNA Ribosomes Serotyping Site Structure Systemic infection Testing Translations Upper Respiratory Infections Viral Virus Virus Diseases Virus Replication body system deep sequencing endoribonuclease fitness inorganic phosphate insight member neonate novel pathogen prevent public health relevance recombinant virus research study tool transcriptome sequencing viral RNA virus host interaction virus pathogenesis

项目摘要

DESCRIPTION (provided by applicant): Picornaviruses replicate via RNA intermediates in the cytoplasm of infected cells, producing dsRNAs that activate antiviral host responses, including the OAS-RNase L pathway. Recently, we discovered a viral RNA molecule that inhibits the endoribonuclease domain of RNase L, and we characterized individual steps of RNA replication, including reiterative transcription mechanisms required for viral RNA polyadenylation. Enterovirus RNA polymerase (3Dpol) is well characterized at the structural level, yet, it is still unclear how conserved amino acid sequences and structures in the polymerase mediate distinct aspects of viral RNA replication. We intend to focus on a dsRNA clamp of 3Dpol that holds RNA products of viral replication as they exit the polymerase. Our preliminary data indicate that mutations in the dsRNA clamp of 3Dpol are maintained in infectious virus; however, these mutations impact the polyadenylation of viral RNA, the fidelity of viral RNA replication, the sensitivity of virus to ribavirin and virus fitness. We expect these mutations will also affect virl RNA recombination. After completing the experiments described in this application, we will better understand the structural features of 3Dpol and the manner in which they function. These studies are important for our understanding of antiviral drugs that target viral polymerases and the mechanisms by which recombinant viruses arise in nature, including recombination among wildtype enteroviruses and poliovirus vaccines. Ribonuclease L (RNase L) is an endoribonuclease associated with antiviral and antibacterial defense, cancer and lifespan. Despite the biological significance of RNase L, the RNAs cleaved by this enzyme are poorly defined. We plan to use novel deep sequencing methods to identify the host and viral RNAs targeted by RNase L and determine the functional effect of cleavage in host and viral RNAs. After completing the experiments described in this application, we will better understand how RNase L exerts its antiviral activity and how viruses overcome the antiviral activity of RNase L. Our study will also reveal how RNase L-dependent cleavage sites in 18S rRNA impact ribosome function; thereby revealing how one molecular target of RNase L impacts multiple phenotypes (antiviral activity, apoptosis, cancer and lifespan). New deep sequencing methods provide unprecedented opportunities to better understand virus replication and pathogenesis. During the previous funding period, we developed and validated cyclic phosphate cDNA synthesis and Illumina sequencing methods which reveal the frequency and location of antiviral endoribonuclease cleavage sites in host and viral RNAs. Other members of the research community developed circle cDNA sequencing to measure the fidelity of viral RNA replication. Together, these two deep sequencing methods provide the means for substantial new insights into the host-virus interactions that determine the outcomes of virus infections. We intend to use these new deep sequencing methods, in conjunction with other well-established experimental conditions, to build upon recent discoveries.

描述（由适用提供）：Picornaviruses通过RNA中间体在感染细胞的细胞质中复制，产生激活抗病毒宿主反应的DSRNA，包括OAS-RNase L途径。最近，我们发现了一种病毒RNA分子，该病毒分子抑制了RNase L的内膜核酸酶结构域，并表征了RNA复制的个体步骤，包括病毒RNA聚腺苷酸化所需的重复转录机制。肠病毒RNA聚合酶（3DPOL）在结构水平上具有很好的特征，但仍不清楚聚合酶培养基中如何组成病毒RNA复制的不同方面的氨基酸序列和结构。我们打算专注于3DPol的DSRNA夹，该夹具在退出聚合酶时保持病毒复制的RNA产物。我们的初步数据表明，3DPol的DSRNA夹中的突变保持在传染病中。然而，这些突变影响病毒RNA的聚腺苷酸化，病毒RNA复制的保真度，病毒对利巴韦林和病毒适应性的敏感性。我们预计这些突变也会影响病毒RNA重组。完成本应用程序中描述的实验后，我们将更好地了解3DPol的结构特征及其功能的方式。这些研究对于我们对靶向病毒聚合酶的抗病毒药物以及重组病毒在自然界中出现的机制的理解非常重要，包括野生型肠病毒和脊髓灰质炎病毒疫苗之间的重组。核糖核酸酶L（RNase L）是一种与抗病毒和抗菌防御，癌症和寿命相关的内核酸酶。尽管RNase L具有生物学性，但该酶裂解的RNA的定义很差。我们计划使用新型的深层测序方法来识别RNase L靶向的宿主和病毒RNA，并确定宿主和病毒RNA中裂解的功能效应。在完成本应用中描述的实验后，我们将更好地了解RNase L如何发挥其抗病毒活性以及病毒如何克服RNase L的抗病毒活性。我们的研究还将揭示RNase L依赖性的裂解位点在18S RRNA撞击核糖体功能中如何依赖RNase L依赖性裂解位点；从而揭示了RNase L的一个分子靶标如何影响多种表型（抗病毒活性，凋亡，癌症和寿命）。新的深层测序方法提供了前所未有的机会，可以更好地了解病毒复制和发病机理。在上一个资金期间，我们开发了并验证了循环磷酸cDNA合成和Illumina测序方法，这些方法揭示了宿主和病毒RNA中抗病毒内切核酸酶切割位点的频率和位置。研究界的其他成员开发了圆cDNA测序，以衡量病毒RNA复制的保真度。总之，这两种深层测序方法为确定病毒感染结果的宿主病毒相互作用提供了大量新见解的手段。我们打算将这些新的深层测序方法与其他公认的实验条件结合使用，以建立在最新发现的基础上。