The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1 alpha (HIF-1 alpha) and HIF-2 alpha were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761-6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1 alpha m (degradation-resistant HIF-1 alpha) caused substantial reduction in HIF-1 alpha-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1 alpha m in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1 alpha. Also, purified HIF-1 alpha and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1 alpha-dependent promoters was attributed to degradation of HIF-1 alpha after binding with ORF34, since the overall amount of wild-type HIF-1 alpha but not the degradation-resistant one (HIF-1 alpha m) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1 alpha in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1 alpha degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1 alpha, leading to its degradation via the proteasome-dependent pathway.