Proteomic analysis of the Kaposi's sarcoma-associated herpesvirus terminal repeat element binding proteins.
Terminal repeat (TR) elements of Kaposi's sarcoma-associated herpesvirus (KSHV), the potential origin sites of KSHV replication, have been demonstrated to play important roles in viral replication and transcription and are most likely also critical for the segregation of the KSHV genome to daughter cells. To search for the cellular proteins potentially involved in KSHV genome maintenance, we performed affinity chromatography analysis, using KSHV TR DNA as the affinity ligand. Proteomic analysis was then carried out to identify the TR-interacting proteins. We identified a total of 123 proteins from both KSHV-positive and -negative cells, among which most were identified exclusively from KSHV-positive cells. These proteins were categorized as proliferation/cell cycle regulatory proteins, proteins involved in spliceosome components, such as heterogeneous nuclear ribonuclear proteins, the DEAD/H family, the switch/sucrose nonfermenting protein family, splicing factors, RNA binding proteins, transcription regulation proteins, replication factors, modifying enzymes, and a number of proteins that could not be broadly categorized. To support the proteomic results, the presence of four candidate proteins, ATR, BRG1, NPM1 and PARP-1, in the elutions was further characterized in this study. The binding and colocalization of these proteins with the TR were verified using chromatin immunoprecipitation and immunofluorescence in situ hybridization analysis. These newly identified TR binding proteins provide a number of clues and potential links to understanding the mechanisms regulating the replication, transcription, and genome maintenance of KSHV. This study will facilitate the generation and testing of new hypotheses to further our understanding of the mechanisms involved in KSHV persistence and its associated pathogenesis.
Si
Huaxin
H
Department of Microbiology and Abramson Comprehensive Cancer Center, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA.
Verma
Subhash C
SC
Robertson
Erle S
ES
D002467:Cell Nucleus / Q000378:metabolism; D002845:Chromatography; D016678:Genome; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D000090243:Nucleophosmin; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D017381:Spliceosomes / Q000378:metabolism; D020079:Terminal Repeat Sequences; D014779:Virus Replication
D008024:Ligands; C000716967:NPM1 protein, human; D000090243:Nucleophosmin; D012313:RNA