GENETIC AND BIOCHEMICAL STUDIES OF THE KSHV LANA GENE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。卡波西肉瘤(KS)相关疱疹病毒(KSHV)与KS、原发性渗出性淋巴瘤和多中心性Castleman？S病有关。KSHV感染主要是潜伏的，双链DNA基因组保持为环状的、染色体外的Episome，从中只表达一部分病毒基因，包括潜伏期相关核抗原1(LANA1)。在有丝分裂过程中，LANA1是病毒基因组复制和分配到后代核所必需的。LANA1激活/抑制细胞启动子。LANA1的C末端250个氨基酸包含LANA寡聚所需的区域和与KSHV末端重复序列(TRDNA)特异结合的区域，并与参与DNA复制和基因表达的多种细胞蛋白相互作用。这个项目的目的是确定这些相互作用的细胞蛋白，这些蛋白可能是LANA1的S扮演各种角色所必需的。在大肠杆菌BL21(DE3)中表达了GST和GST-LANA(C-末端)融合表达载体，并用GST琼脂糖亲和层析柱进行分离。每个分离株都与来自BJAB细胞的提取物孵育，BJAB细胞是KSHV阴性的B淋巴细胞株。分离蛋白经GST亲和层析后，1D-SDS-PAGE分离，凝胶内胰酶消化。使用Bruker Reflex IV质谱仪进行MALDI-TOF质谱分析。串联MS使用应用生物系统Q-Star Pulsar I QoTOF MS或自制的装有用于IRMPD的CO2(10.6 mm)激光的振动冷却MALDI-FTMS系统进行。使用Mascot(Matrix Science)和Aldente(SwissProt)数据库搜索引擎对数据进行分析。通过对初步的质谱学数据的研究，初步鉴定了至少一种被认为与DNA复制和基因表达有关的细胞蛋白质。然而，由于缺乏GST-LANA1融合蛋白的证据，因此需要重复实验程序。对LANA1和随后的复杂洗脱液使用替代融合构建体表达的进一步研究目前也在调查中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kaposi's Sarcoma (KS)-associated herpesvirus (KSHV) is linked with KS, primary effusion lymphoma, and multicentric Castleman?s disease. KSHV infection is predominantly latent, with the double-stranded DNA genome maintained as a circular, extrachromosomal episome from which only a subset of viral genes is expressed, including latency-associated nuclear antigen 1 (LANA1). LANA1 is required for both replication and partitioning of viral genomes to progeny nuclei during mitosis. LANA1 activates/ represses cellular promoters. The C-terminus 250 amino acids of LANA1 contain regions required for LANA oligomerization and specific binding to KSHV terminal repeat (TR) DNA and interacts with several cellular proteins involved in DNA replication and gene expression. This project aims to identify these interacting cellular proteins that may be required for LANA1's various roles. GST and GST-LANA(C-terminal) fusion constructs were expressed in E. coli BL21(DE3) cells and isolated using a GST Sepharose affinity column. Each isolate was incubated with extract from BJAB cells, a KSHV-negative B-lymphocyte cell line. After GST affinity-based separation, isolated proteins were separated by 1D-SDS-PAGE and subjected to in-gel tryptic digestion. MALDI-TOF mass spectrometry was performed using a Bruker Reflex IV mass spectrometer. Tandem MS was performed using either an Applied Biosystems Q-Star Pulsar I QoTOF MS, or a home-built Vibrational Cooling MALDI- FTMS system fitted with a CO2 (10.6 mm) laser for IRMPD. Data were analysed using Mascot (Matrix Science) and Aldente (SwissProt) database search engines. Investigation of preliminary mass spectrometric data enabled tentative identification of at least one cellular protein believed to be involved in DNA replication and gene expression. The lack of evidence of the GST-LANA1 fusion protein, however, has required the experimental procedure to be repeated. Further studies into the use of alternative fusion constructs for the expression of LANA1 and subsequent complex elutions are also currently under investigation.