Role of Angiogenin in KSHV Biology and Pathogenesis

In this study we show that LANA-1 and angiogenin (ANG) colocalize and co-immunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) and TIVE-LTC cells. LANA-1 and ANG interaction occurred in the absence of KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG co-eluted with LANA-1, p53 and Mdm2 in high molecular weight fractions, and LANA-1, p53 and Mdm2 also co-immunoprecipitated with ANG. LANA-1, ANG and p53 colocalized in KSHV infected cells and colocalization between ANG and p53 was also observed in LANA-1 negative cells. Deletion constructs of ANG suggested that C-terminal 104-123 amino acid region is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53 and LANA-1-p53, induction of p53, p21 and Bax proteins, increased cytoplasmic localization of p53, down-regulation of Bcl-2, increased cleavage of caspase-3 and apoptosis of cells. No such effects were observed in KSHV negative BJAB cells. Phosphorylation of p53 at serine 15 that is essential for p53 stabilization and for p53's apoptotic and cell cycle regulation functions was increased in sh-ANG transduced BCBL-1 cells. Together, these studies suggest that the anti-apoptosis observed in KSHV infected cells and suppression of p53 functions could in part be mediated by ANG and KSHV has probably evolved to utilize angiogenin's multiple functions for the maintenance of its latency and cell survival. Thus targeting ANG to induce apoptosis of cells latently infected with KSHV is a potential attractive therapeutic strategy against KSHV infection and associated malignancies.;We also carried out mass spectrometric analyses of TIVE-LTC proteins immunoprecipitated by anti-LANA-1 and anti-ANG antibodies to identify cellular proteins that associate with both ANG and LANA-1. Our analyses identified twenty-eight common cellular proteins such as ribosomal proteins, structural proteins, t-RNA synthetases, metabolic pathway enzymes, chaperons, transcription factors, anti-oxidants, and ubiquitin proteosome proteins. LANA-1 and ANG interaction with one of the proteins, annexin A2, was validated. Annexin A2 has been shown to play roles in cell proliferation, apoptosis, plasmin generation, exocytosis, endocytosis, and cytoskeleton reorganization. It is also known to associates with glycolytic enzyme 3-phosphoglyceratekinase in the primer recognition protein (PRP) complex that interacts with DNA polymerase &agr; in the lagging strand of DNA during replication. A higher level of annexin A2 is expressed in KSHV (+), but not in EBV (+), B lymphoma cell lines. Annexin A2 colocalized with several LANA-1 punctate spots in KSHV (+) body-cavity B-cell lymphoma (BCBL-1) cells. In triple staining analyses, we observed annexin A2-ANG-LANA-1, annexin A2-ANG and ANG-LANA-1 colocalizations. Annexin A2 appeared as punctate nuclear dots in LANA-1 positive TIVE-LTC cells. In LANA-1 negative TIVE-LTC cells, annexin A2 was detected predominately in the cytoplasm with some nuclear spots and colocalization with ANG was observed mostly in the cytoplasm. Annexin A2 co-immunoprecipitated with LANA-1 and ANG in TIVE-LTC and BCBL-1 cells, and with ANG in 293T cells independent of LANA-1. This suggested that annexin A2 forms a complex with LANA-1 and ANG as well as a separate complex with ANG. Silencing annexin A2 in BCBL-1 cells resulted in significant cell death, down-regulation of cell cycle associated Cdk6, cyclin D, E and A proteins, and down-regulation of LANA-1 and ANG expression. No effect was seen in KSHV (-) lymphoma (BJAB and Ramos) and 293T cells. These studies suggest that LANA-1 association with annexin A2/ANG could be more important than ANG association with annexin A2, and KSHV is probably using annexin A2 to maintain the viability and cell cycle regulation of latently infected cells.;Overall my studies highlight the importance of angiogenin in KSHV infected cells and presents it as an indispensible protein for KSHV. Since the identified LANA-1 and ANG interacting common cellular proteins are hitherto unknown to KSHV and ANG biology, this offers a starting point for further analysis of their roles in KSHV biology which may lead to identification of potential therapeutic targets to control KSHV latency and associated malignancies. (Abstract shortened by UMI.).