Identifying novel activities of KSHV/HHV8 DNA replication proteins

鉴定 KSHV/HHV8 DNA 复制蛋白的新活性

• 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
• 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

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%的人类癌症的病原体。最近发现的被 卡波西肉瘤疱疹病毒（KSHV/HHV 8）已知引起三种人类癌症。有效 治疗KSHV需要抗病毒治疗剂。病毒DNA复制和KSHV DNA复制 蛋白质是成功的病毒复制所必需的，因此是有吸引力的治疗靶点。的重点 该提案描述了KSHV核心DNA复制蛋白的功能和分子相互作用， 以便开发针对KSHV感染的潜在治疗策略。以前的研究使用序列 KSHV和相关疱疹病毒之间的同源性，以确定六种基本蛋白质的保守蛋白质功能 由KSHV编码的核心DNA复制蛋白，ORF 6（SSB），ORF 9（POL），ORF 40/41（PAF），ORF 44（HEL）， ORF 56（PRI）、ORF 59（PF）和起始结合蛋白ORF 50（RTA）。然而，蛋白质序列同源性 仅在20-50%范围内，因此预测蛋白质功能较差;因此， 和深入的生化分析的个别KSHV病毒DNA复制蛋白，或在组合， 需要确定其全部功能（目标1）。为了评价体外蛋白活性，高分辨率 电子显微镜（EM）将补充生化分析。通过EM直接可视化产生 定性数据（异质蛋白质复合物、寡聚状态、DNA结构）和定量数据 (DNA蛋白质结合位置作图、分子大小比较）。我们的初步发现提供了新的 深入了解KSHV蛋白的活性。本提案的第一个目的将表征分子 已经纯化的KSHV DNA复制蛋白（ORF 6，ORF 9，ORF 44， ORF 59和ORF 50）。这些发现将为KSHV复制提供有价值的见解，并为未来的研究提供信息。 从人细胞培养系统中纯化的蛋白质，以直接比较病毒蛋白质的影响， 由昆虫细胞产生的蛋白质的翻译修饰与人细胞天然修饰。的 该提议的第二个目的集中于开发用于在生理学上产生病毒蛋白的系统。 相关的人类细胞系。我们以前已经使用一种新的方法生产了七种KSHV复制蛋白中的五种。 昆虫Sf 9细胞系统，但商业和实验室尝试表达和纯化其余两种蛋白质 ORF 40/41和ORF 54的基因组克隆尚未成功。我们假设通过利用 相关的人类细胞类型，我们将克服在非人类细胞系中生产病毒蛋白的挑战 并增强纯化蛋白的功能性（目的2）。对核心DNA的深入分子研究 复制蛋白将促进对KSHV生物学和γ-疱疹病毒复制的一般理解 从该提案中产生的数据将为未来旨在确定 病毒特异性抑制剂，以防止KSHV感染。