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.
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