RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus known to cause several tumors and hyperproliferative disorders. While there has been reports of KSHV activating and increasing hypoxia-inducible factors (HIFs), this is the first report investigating and establishing the extent to which KSHV has evolved to reproduce the effects of hypoxia. We demonstrate that the cellular changes in gene expression induced by KSHV infection include many of the changes induced by hypoxia. This has substantial implications for the biology of KSHV and the pathogenesis of KSHV-associated cancers. To achieve this, we used mRNA-sequencing and small RNA-sequencing in combination with bioinformatics analysis, and orthogonal assays such as qRT-PCR and Taqman assays to determine the effects of hypoxia on miRNA and mRNA expression. We showed that not only was there a 34% overlap between the hypoxic response and KSHV infection, but also that miRNA miR-210, a HIF-target known to have anti-apoptotic, angiogenic, and oncogenic properties, was independently and additively increased by KSHV infection and hypoxia. Furthermore, we explored the effects of hypoxia on KSHV miRNAs and consistently observed that none of the KSHV miRNAs are affected by oxygen deprivation. These studies suggest that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Previous studies have shown that hypoxia and HIFs activate KSHV-encoded genes, including several involved in tumor formation. These findings suggest that targeting HIFs or hypoxia pathways could block this positive feedback loop between KSHV and hypoxia and thus might be a useful strategy to treat KSHV-related tumors or other diseases. We believe these are important findings with broad implication for the understanding of the biology of KSHV and other oncoviruses and the pathogenesis of KSHV-induced tumors. As such, it should be of interest to the broad community of investigators and clinicians interested in the biology of oncoviruses, virus-induced cellular changes, and the pathogenesis and therapy of virus-induced tumors.