Identifying novel activities of KSHV/HHV8 DNA replication proteins

鉴定 KSHV/HHV8 DNA 复制蛋白的新活性

基本信息

批准号：
10359791
负责人：
Lindsey M Costantini
金额：
$ 14.8万
依托单位：
NORTH CAROLINA CENTRAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-15 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10359791
关键词：
Address Amino Acid Sequence Homology Antiviral Agents Architecture Binding Binding Proteins Binding Sites Biochemical Biology Cell Culture System Cell Culture Techniques Cell Line Cells Closure by clamp Complement Complex Conflict (Psychology)DNA DNA Binding DNA Polymerase Inhibitor DNA Sequence DNA biosynthesis DNA mapping DNA replication fork DNA-Directed DNA Polymerase Data Electron Microscopy Engineering Foundations Frequencies Future Gel Genetic Transcription Goals HIV HIV Envelope Protein gp120 Herpesviridae Human Human Cell Line Human Herpesvirus 8 Image In Vitro Individual Infection Insect Proteins Insecta Kaposi Sarcoma Legal patent Location Malignant Neoplasms Mammalian Cell Measurement Methods Modeling Modification Molecular Molecular Conformation Multicentric Angiofollicular Lymphoid Hyperplasia Phosphorylation Phosphorylation Site Physiological Positioning Attribute Post-Translational Protein Processing Production Protein Conformation Proteins Publications Publishing Replication Origin Reporting Resolution Sequence Homology Slide Specificity Structure System Technology Therapeutic Translations Vaccines Variant Viral Viral Proteins Virus Virus Replication Visualization base cell type comparative dimer effective therapy experimental study gammaherpesvirus inhibitor insight molecular size monomer mutant nanoscale new therapeutic target novel preservation prevent primary effusion lymphoma protein complex protein expression protein function protein transport reconstitution therapeutic target vector viral DNA

Address Amino Acid Sequence Homology Antiviral Agents Architecture Binding Binding Proteins Binding Sites Biochemical Biology Cell Culture System Cell Culture Techniques Cell Line Cells Closure by clamp Complement Complex Conflict (Psychology)DNA DNA Binding DNA Polymerase Inhibitor DNA Sequence DNA biosynthesis DNA mapping DNA replication fork DNA-Directed DNA Polymerase Data Electron Microscopy Engineering Foundations Frequencies Future Gel Genetic Transcription Goals HIV HIV Envelope Protein gp120 Herpesviridae Human Human Cell Line Human Herpesvirus 8 Image In Vitro Individual Infection Insect Proteins Insecta Kaposi Sarcoma Legal patent Location Malignant Neoplasms Mammalian Cell Measurement Methods Modeling Modification Molecular Molecular Conformation Multicentric Angiofollicular Lymphoid Hyperplasia Phosphorylation Phosphorylation Site Physiological Positioning Attribute Post-Translational Protein Processing Production Protein Conformation Proteins Publications Publishing Replication Origin Reporting Resolution Sequence Homology Slide Specificity Structure System Technology Therapeutic Translations Vaccines Variant Viral Viral Proteins Virus Virus Replication Visualization base cell type comparative dimer effective therapy experimental study gammaherpesvirus inhibitor insight molecular size monomer mutant nanoscale new therapeutic target novel preservation prevent primary effusion lymphoma protein complex protein expression protein function protein transport reconstitution therapeutic target vector viral DNA

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项目摘要

Project Summary Viruses are the causative agents of approximately 12% of human cancers. The most recently discovered herpesvirus, Kaposi’s sarcoma herpesvirus (KSHV/HHV8) is known to cause three human cancers. Effective antiviral therapeutics are needed for the treatment of KSHV. Viral DNA replication and the KSHV DNA replication proteins are essential to successful viral replication and, thus, appealing therapeutic targets. The focus of this proposal is characterizing the functions and molecular interactions of the core KSHV DNA replication proteins in order to develop potential therapeutic strategies against KSHV infection. Previous studies used sequence homology between KSHV and related herpesviruses to determine conserved protein functions of the six essential core DNA replication proteins encoded by KSHV, ORF6(SSB), ORF9(POL), ORF40/41(PAF), ORF44(HEL), ORF56(PRI), ORF59(PF) and the origin binding protein, ORF50(RTA). However, protein sequence homologies only range from 20-50% and thus poorly predict protein function; therefore, an ultrastructural characterization and in-depth biochemical analysis of individual KSHV viral DNA replication proteins, or in combinations, is needed to identify their full range of functions (Aim 1). To evaluate the in vitro protein activities, high resolution electron microscopy (EM) will be complemented by biochemical analysis. Direct visualization via EM produces qualitative data (heterogeneous protein complexes, oligomeric state, DNA architecture) and quantitative data (DNA mapping of protein binding locations, molecular size comparisons). Our preliminary finding provide novel insights into the activities of KSHV proteins. The first aim of this proposal will characterize the molecular interactions and activities of a subset of already purified KSHV DNA replication proteins (ORF6, ORF9, ORF44, ORF59 and ORF50). The findings will provide valuable insights into KSHV replication and inform future studies of proteins purified from a human cell culture system to directly compare the impact of viral protein post- translational modifications of proteins produced from insect cells with human cell native modifications. The second aim of this proposal is focused on developing a system for generating viral proteins in physiologically relevant human cell lines. We have previously produced five of the seven KSHV replication proteins using an insect Sf9 cell system, but commercial and lab attempts to express and purify the remaining two proteins (ORF40/41 and ORF54) from non-mammalian cells have been unsuccessful. We hypothesize that by utilizing relevant human cell types, we will overcome the challenges of producing viral proteins in non-human cell lines and enhance the functionality of the purified proteins (Aim 2). This in-depth molecular study of the core DNA replication proteins will advance the general understanding of KSHV biology and gamma-herpesvirus replication and the data generated from this proposal will provide the foundation for future proposals aimed at identifying virus specific inhibitors to prevent KSHV infection.

项目摘要病毒是大约12％的人类癌症的严重药物。最近发现的疱疹病毒，卡波西的肉瘤疱疹病毒（KSHV/HHV8）众所周知会引起三种人类癌症。有效的需要抗病毒疗法来治疗KSHV。病毒DNA复制和KSHV DNA复制蛋白质对于成功的病毒复制至关重要，因此是出现治疗靶标的。重点提案正在表征核心KSHV DNA复制蛋白的功能和分子相互作用为了制定针对KSHV感染的潜在治疗策略。以前的研究使用了序列 KSHV和相关疱疹病毒之间的同源性，以确定六个必需的六个必需蛋白质功能由KSHV，ORF6（SSB），ORF9（POL），ORF40/41（PAF），ORF44（HEL），ORF9（pol）编码的核心DNA复制蛋白，ORF6（SSB），ORF9（POL）， ORF56（PRI），ORF59（PF）和原点结合蛋白，ORF50（RTA）。但是，蛋白质序列同源性仅在20-50％范围内预测蛋白质功能。因此，超微结构特征对单个KSHV病毒DNA复制蛋白或组合中的单个KSHV病毒DNA复制蛋白的深入生化分析是需要确定其全部功能范围（AIM 1）。为了评估体外蛋白质活性，高分辨率电子显微镜（EM）将通过生化分析完成。通过EM Productions直接可视化定性数据（异源蛋白质复合物，低聚状态，DNA结构）和定量数据（蛋白质结合位置的DNA映射，分子大小比较）。我们的初步发现提供了小说洞悉KSHV蛋白的活性。该提案的第一个目的是表征分子已经纯化的KSHV DNA复制蛋白（ORF6，ORF9，ORF44， ORF59和ORF50）。这些发现将为KSHV复制提供宝贵的见解，并为未来的研究提供信息从人类细胞培养系统中纯化的蛋白质的直接比较病毒蛋白的影响由人类细胞天然修饰产生的昆虫细胞产生的蛋白质的翻译修饰。这该提案的第二个目的是开发一种在物理上生成病毒蛋白的系统相关的人类细胞系。我们以前已经使用一个昆虫SF9细胞系统，但商业和实验室试图表达和净化其余两种蛋白质（ORF40/41和ORF54）来自非哺乳动物细胞的未成功。我们通过使用相关的人类细胞类型，我们将克服在非人类细胞系中产生病毒蛋白的挑战并增强纯化蛋白质的功能（AIM 2）。这项对核心DNA的深入分子研究复制蛋白将提高对KSHV生物学和伽马病毒复制的一般理解从本提案中产生的数据将为未来的建议奠定基础，以识别病毒特异性抑制剂可预防KSHV感染。