Proteomic Profiling Identifies Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Encoded LANASIM-Associated Proteins in Hypoxia

Hypoxia signaling is a key regulator in the development and progression of many types of human malignancies, including viral cancers. The latency-associated nuclear antigen (LANA), encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) during latency, is a multifunctional protein that plays an essential role in viral episome maintenance and lytic gene silencing for inducing tumorigenesis. Although our previous studies have shown that LANA contains a SUMO-interacting motif (LANA(SIM)), and hypoxia reduces SUMOylated KAP1 association with LANA(SIM), the physiological proteomic network of LANA(SIM)-associated cellular proteins in response to hypoxia is still unclear. In this study, we individually established cell lines stably expressing wild-type LANA (LANAWT) and its SIM-deleted mutant (LANA(dSIM)) and treated them with or without hypoxia, followed by coimmunoprecipitation and mass spectrometry analysis to systemically identify the hypoxia-responsive profile of LANA(SIM)-associated cellular proteins. We found that in hypoxia, the number of cellular proteins associated with LANAWT instead of LANAd(SIM) was dramatically increased. Functional network analysis revealed that two major pathways, which included cytoskeleton organization and DNA/RNA binding and processing pathways, were significantly enriched for 28 LANA(SIM)-associated proteins in response to hypoxia. HNRNPU was one of the proteins consistently identified that interacted with LANA(SIM) in different proteomic screening systems and responded to hypoxia. This study provides a proteomic profile of LANA(SIM)-associated proteins in hypoxia and facilitates our understanding of the role of the collaboration between viral infection and the hypoxia response in inducing viral persistence and tumorigenesis. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) has been reported to be involved in the regulation of host proteins in response to hypoxic stress. LANA, one of the key latent proteins, contains a SUMO-interacting motif (LANA(SIM)) and reduces the association with SUMOylated KAP1 upon hypoxic treatment. However, the physiological systematic network of LANA(SIM)-associated cellular proteins in hypoxia is still unclear. Here, we revealed two major pathways, which included cytoskeleton organization and DNA/RNA binding and processing pathways, that were significantly enriched for 28 LANA(SIM)-associated proteins in hypoxia. This discovery not only provides a proteomic profile of LANA(SIM)-associated proteins in hypoxia but also facilitates our understanding of the collaboration between viral infection and hypoxic stress in inducing viral persistence and tumorigenesis.