title: The Kaposi's sarcoma-associated herpesvirus ORF34 protein binds to HIF-1α and causes its degradation via the proteasome pathway.
abstract: 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 1α (HIF-1α) and HIF-2α 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-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway.
authors.affiliation: Division of Biotechnology and Molecular Medicine and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA. mhaque@agcenter.lsu.edu
authors.forename: Muzammel
authors.initials: M
authors.lastname: Haque
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Kousoulas
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis; D019288:Herpesvirus 8, Human / Q000472:pathogenicity*; D054884:Host-Pathogen Interactions; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D047468:Immunoprecipitation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D059748:Proteolysis; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014764:Viral Proteins; D046988:Proteasome Endopeptidase Complex