Kaposi's sarcoma-associated herpesvirus (KSHV)
infection and
latency-associated nuclear antigen (LANA-1) upregulate the multifunctional
protein angiogenin (ANG). Our studies demonstrate that silencing ANG or inhibiting its nuclear translocation downregulates KSHV LANA-1 expression and ANG is necessary for KSHV latency, anti-apoptosis and angiogenesis (Sadagopan et al., J. Virol. 83:3342-3364, 2009; Sadagopan et al., J Virol. 85:2666-2685, 2011). Here we show that LANA-1 interacts with ANG and colocalizes in latently infected endothelial
telomerase-immortalized human umbilical vein endothelial (TIVE-LTC) cells. Mass spectrometric analyses of TIVE-LTC
proteins immunoprecipitated by anti-LANA-1 and ANG
antibodies identified 28 common cellular
proteins such as
ribosomal proteins, structural
proteins,
tRNA synthetases, metabolic pathway
enzymes, chaperons,
transcription factors,
antioxidants, 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 associate with glycolytic
enzyme 3-phosphoglyceratekinase in the primer recognition
protein (PRP) complex that interacts with
DNA polymerase α in the lagging strand of
DNA during replication. A higher level of
annexin A2 is expressed in KSHV+ but not in Epstein-Barr virus (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 coimmunoprecipitated 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, downregulation of cell cycle-associated Cdk6 and of
cyclin D, E, and A
proteins, and downregulation 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 probably uses
annexin A2 to maintain the viability and cell cycle regulation of latently infected cells. 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.