Proteomic Profiling Identifies Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Encoded LANA^SIM-Associated Proteins in...

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 (LANA^WT) 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 LANA^WT instead of LANA^dSIM 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.