The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Interacts and Stabilizes HIF-2α via Binding to the HIF-2α bHLH and PAS Domains

J Virol. 2019 Aug 13;93(17):e00764-19. doi: 10.1128/JVI.00764-19. Print 2019 Sep 1.

Abstract

Hypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposi's sarcoma (KS) and other AIDS-related malignancies. Kaposi's sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1α and HIF-2α. HIF-1α and HIF-2α have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1α and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1α-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2α in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2α and that both bHLH and PAS domains of HIF-2α interacted with ORF34. Unlike HIF-1α, dose-dependent coexpression of ORF34 stabilized the HIF-2α protein, ensuring HIF-2α-dependent transcriptional activity. The ORF34 protein enhanced HIF-2α ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1α and HIF-2α proteins.IMPORTANCE Hypoxia inducible factor 1α (HIF-1α) and HIF-2α are transcription factors which play important roles in the Kaposi's sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2α, in contrast to its role in binding HIF-1α and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1α and HIF-2α expression.

Keywords: Kaposi’s sarcoma-associated herpesvirus; hypoxia inducible factors (HIFs); ubiquitination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / chemistry*
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Binding Sites
  • Capsid Proteins / chemistry*
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism*
  • Gene Deletion
  • Gene Expression Regulation, Viral
  • HEK293 Cells
  • Herpesvirus 8, Human / genetics
  • Herpesvirus 8, Human / metabolism
  • Herpesvirus 8, Human / physiology*
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • Life Cycle Stages
  • Protein Domains
  • Protein Stability
  • Transcription, Genetic
  • Ubiquitination

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • Capsid Proteins
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • ORF38 protein, Kaposi's sarcoma-associated herpesvirus
  • endothelial PAS domain-containing protein 1