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

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 theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted 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.IMPORTANCEHypoxia 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 theORF34gene is involved in the regulation of KSHV lytic gene expression, since deletion ofORF34resulted 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.

Kaposi’s sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis

“…Herein, we show that upon KSHV lytic switch, HIF2a levels and subcellular localization are regulated to activate the alternative eIF4E2 translation initiation of viral mRNAs. It was recently shown that the KSHV lytic protein ORF34 is involved in HIF2a stabilization (Haque and Kousoulas, 2019). However, because in our system ORF34 has late early lytic expression, we suspect that other KSHV-related mechanisms are involved in the upregulation of HIF2a occurring during lytic reactivation (Figure 1; Figure S1).…”

The Zebrafish Xenograft Platform—A Novel Tool for Modeling KSHV-Associated Diseases

“…Remarkably, the transcriptome of KSHV latently infected cells resembles a hypoxic gene expression signature, possibly due to the actions of the latent protein LANA that increases levels HIF-1α mRNA and protein [44], suggesting that HRE activity in infected cells could be influenced by products of the latent transcriptome, as well as by environmental oxygen. Later during lytic replication, the ORF34 lytic protein binds and stabilizes HIF-2α [45], which could promote eIF4E2-dependent translation [27]. Interestingly, HIF-1α is required for normal lytic gene expression during normoxia in both KSHV and murine gammaherpesvirus 68 infections [46,47].…”

Hypoxia and hypoxia‐inducible factors in Kaposi sarcoma‐associated herpesvirus infection and disease pathogenesis

“…In this regard, it has been recently discovered that the ORF34 gene product, which can be upregulated by hypoxia or HIF, can stabilize HIF-2α while at the same time cause the degradation of HIF-1α. 56,57 Deletion of ORF34 from the KSHV genome resulted in decreased early gene expression and blocked late gene expression.…”

“…At least one KSHV protein, the ORF34 gene product, is known to increase degradation of HIF-1α while stabilizing HIF-2α. 56,57 Another KSHV protein, vCyclin, also leads to degradation of HIF-1α at certain stages of infection to allow for cell cycle progression but what is its effect on HIF-2α? In some cases, the cells being examined may not express HIF-2α so the point may be moot.…”