Protein complexes associated with the Kaposi's sarcoma-associated herpesvirus-encoded LANA

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA is known to modulate viral and cellular gene expression. We report here on some initial proteomic studies to identify cellular proteins associated with the amino and carboxy-terminal domains of LANA. The results of these studies show an association of known cellular proteins which support LANA functions and have identified additional LANA-associated proteins. These results provide new evidence for complexes involving LANA with a number of previously unreported functional classes of proteins including DNA polymerase, RNA helicase and cell cycle control proteins. The results also indicate that the amino terminus of LANA can interact with its carboxy-terminal domain. This interaction is potentially important for facilitating associations with other cell cycle regulatory proteins which include CENP-F identified in association with both the amino and carboxy-termini. These novel associations add to the diversity of LANA functions in relation to the maintenance of latency and subsequent transformation of KSHV infected cells.

Figure 1

GST tagged LANA-amino terminal domain and GST tagged LANA-carboxy terminal domain bound on Glutathione Sepharose beads was used as bait to pull down interacting protein from BC3 cell nuclear extract. BC-3 nuclear extract (500μg) was first pre-cleared with GST only (6mg) by incubating at 4°C for 4 hours with rotation. The supernatant was then pre-cleared with GST-2TK for 2 more hours at 4°C in similar way. The supernatant from this step was then incubated with GST-LANA-N at 4°C overnight. The beads were washed four times with binding buffer by centrifugation at 4°C. The beads were then collected and boiled at 95°C in SDS-PAGE loading buffer and run on SDS-PAGE with gel with appropriate controls. Protein bands unique to LANA pulldown lanes were excised and processed for MALDI-TOF peptide analysis

Figure 2

Proportional distribution of LANA-amino terminal interacting proteins in classes based on their functions. The proteins have been classified based on their molecular function as per the scheme followed by Human protein reference database Copyright © Johns Hopkins University and the Institute of Bioinformatics

Figure 3

Proportional distribution of LANA-carboxy terminal interacting proteins in classes based on their functions. The proteins have been classified based on their molecular function as per the scheme followed by Human protein reference database Copyright © Johns Hopkins University and the Institute of Bioinformatics.

Figure 4

CENP-F interacts with LANA. 293T were transected with LANA-Myc expressing construct and harvested after 24 hours. CENP-F was immuno-precipitated from the cell lysate using specific antibody and then probed for coimmunoprecipitated LANA with anti-Myc antibody. Anti-p53 antibody was used as positive control to coimmunoprecipitate LANA and normal goat serum was used as non-specific antibody control. Lane 2 (fig. 5a, top panel) shows the coimmunoprecipated LANA compared to 10% input in lane 1. Lane 3 shows the coimmunoprecipitated LANA along with p53 as positive control. Bottom panel (fig 5a) shows the same membrane reprobed with CENP-F antibody. The immunofluoroscence assay was done to visualize the localization of LANA (nuclear) and CENP-F (nuclear membrane) in KSHV positive BCBL-1 cells. The cells were blocked in G2/M phase by nocadozole treatmen (fig. 5b) Panel c shows the localisation of CENP-F in nuclear matrix partially colocalising with LANA. Panel d shows a cell in late G2 phase with CENP-F localising mainly around nuclear envelope

Figure 5

LANA-amino terminus interacts with LANA-carboxy terminal doamin. GST tagged LANA-amino domain (aa 1-340) protein was incubated with in vitro translated LANA-carboxy terminus labeled with S³⁵ Methionine. LANA-carboxy doman showed binding to the amino terminal domain (lower panel, lane 5) but not to GST control (Lower panel, lane 4). The coomassie stained gel with GST-LANA amino terminus is shown in the upper panel.