Proteomic profiling identifies the SIM-associated complex of KSHV-encoded LANA

The Kaposi's sarcoma-associated herpesvirus (KSHV) encoded latent nuclear antigen latency-associated nuclear antigen (LANA) plays an essential role in viral episome maintenance. LANA also contributes to DNA replication and tumorigenesis during latency. Recent studies suggested that LANA was involved in regulation of SUMOylation, which results in chromatin silencing. To examine the pleiotropic effects of LANA protein on host cell gene expression, we utilized MS analysis to identify cellular proteins associated with the small ubiquitin related modifier (SUMO) interacting motif of LANA (LANA(SIM)). In addition to the six bands identified as substantially associated with LANA(SIM), 151 proteins were positively identified by MS/MS analysis. Compared with previous proteomic analysis of the N- and C-truncated mutants of LANA (LANA(NC)), our results revealed that a complex of specific proteins with relatively high SUMOylation and SIM motifs is associated with LANA(SIM). Intriguingly, consistent with our previous report that identified KAP1 as a key component, the in vitro SUMO-2-modified isoform has a substantially higher affinity with LANA(SIM) than the SUMO-1-modified isoform. Moreover, via cluster and pathway analysis, we proposed a hypothetical model for the LANA(SIM) regulatory circuit involving aberrant SUMOylation of cell cycle (particular mitotic), DNA unwinding and replication, and pre-mRNA/mRNA processing related proteins. This study provides a SUMOylated and non-SUMOylated proteome profile of LANA(SIM) -associated complex and facilitates our understanding that viral-mediated gene regulation through SUMOylation is important for Kaposi's sarcoma-associated herpesvirus persistence and pathogenesis.