Date Published: January 3, 2017
Publisher: Public Library of Science
Author(s): Coralie Viollet, David A. Davis, Shewit S. Tekeste, Martin Reczko, Joseph M. Ziegelbauer, Francesco Pezzella, Jiannis Ragoussis, Robert Yarchoan, Paul M. Lieberman.
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 the etiologic agent for several hyperproliferative disorders and tumors, including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and a form of multicentric Castleman disease (MCD) [1–4]. Like other herpesviruses, KSHV has two patterns of gene expression: latent, in which only a small subset of genes are expressed; and lytic, in which the full repertoire of genes are expressed and viral progeny are produced . A number of recent studies have shown that hypoxia and hypoxia-inducible factors (HIFs) are important in the KSHV life cycle and the pathogenesis of KSHV-induced diseases [6–8]. Two of the tumors caused by KSHV, KS and PEL, preferentially arise in relatively hypoxic environments: the extremities and pleural effusions, respectively [9,10]. Cells respond to hypoxic environments by a rapid up-regulation in their levels of two main HIFs, HIF-1 and HIF-2, which in turn enter the nucleus and activate HIF-responsive genes by binding to hypoxia response elements (HRE) in their promoter regions [11,12]. Hypoxia and HIFs can also up-regulate levels of the cellular microRNA (miRNA), miR-210, which in turn affects a number of target genes to promote adaptation to hypoxia [13,14].
Viruses, and especially those that cause chronic infections, are attuned to and respond to changes in their target cells. At the same time, viral infection leads to a number of changes in these target cells, some mediated by the virus and others as part of the host response to infection. Previous studies have shown that hypoxia and HIFs can affect KSHV biology and KSHV-induced tumor formation [6,7,59]. Moreover, KSHV infection and LANA, a KSHV-encoded latent gene, have been shown to induce the up-regulation of HIF  and at least one HIF-responsive gene (VEGFR1) in newly infected endothelial cells . Also, several HIF-responsive genes related to angiogenesis or metabolism have been shown to be elevated in immortalized HUVECs chronically infected with KSHV . However, these data do not provide a complete picture of the cellular response to KSHV infection and the role that hypoxia plays. To further investigate these relationships, we used RNA-Seq analysis to assess the changes induced by hypoxia in SLK cells and compare them with the changes we had previously found in chronically KSHV-infected SLK cells (the SLKK line) ; this paper represents the first global survey of these effects. We found that there was a substantial overlap in the alteration in gene expression induced by KSHV infection and hypoxia. In particular, more than a third (34%) of the genes seen differentially expressed under hypoxia were similarly up- or down-regulated by KSHV latent infection. Also, a majority of the 155 genes that made our cut-off for up-regulation by hypoxia but not for KSHV (Fig 2A) demonstrated a trend towards up-regulation in KSHV infection (Fig 2B). By contrast, only 5 genes up-regulated by hypoxia showed a down-regulation in KSHV-infected cells. Looking at this from the perspective of KSHV-modulated genes, 11% of these genes are genes modulated in the same way by hypoxia. Overall, these results provide evidence that KSHV commandeers the cell response to hypoxia and that hypoxia-related changes in gene expression comprise a substantial portion of the response to KSHV infection. While levels of HIF-1 were greater in hypoxia-exposed SLKK cells than hypoxia-exposed SLK cells, it was noteworthy that a hypoxic signature was observed in normoxic SLK cells in spite of the fact that we could not detect elevated levels of HIF-1 protein. Further studies would be required to determine to which degree the overlap between the hypoxic response and the KSHV infection is due to KSHV-induced up-regulation of HIF, especially under normoxic conditions.