Like other oncogenic viruses, KSHV, the causative agent of Kaposi's sarcoma (KS), the most common malignancy that occurs in patients with AIDS, establishes a latent infection in host cells. Most KS cells are latently infected and only express a limited number of genes, along with a cluster of viral-encoded miRNAs, which have previously been shown to be closely involved in controlling viral replication and latency. Yogev et al. examined the effects of expressing the miRNA cluster and known latency proteins on glucose metabolism in lymphatic endothelial cells (LECs) using lentivirus expression vectors. No metabolic changes were observed with any of the latency proteins tested but expressing the miRNA cluster led to a range of effects that were consistent with induction of the Warburg effect, including reduced consumption of O2, increased secretion of lactate and increased glucose uptake, confirming that KSHV miRNAs have a profound effect on host cell energy metabolism. As oxidative phosphorylation is driven by mitochondria, the authors assessed whether KSHV miRNAs had any effect on mitochondrial function. Using MitoTracker, a fluorescent dye that labels mitochondria in live cells, and qPCR analysis, they found that expression of the miRNA cluster decreased the number of mitochondria present in LECs.
How might the miRNAs mediate this energetic switch? The host protein hypoxia inducible factor 1α (HIF1α) is a master regulator of host cell metabolism and a known mediator of the Warburg effect. Previous work had shown that during latency, KSHV activates HIF1α. The authors therefore assessed whether HIF1α was involved in the KSHV miRNA-mediated metabolic shift. Western blotting revealed that in LECs expressing the miRNA cluster, HIF1α was over-expressed compared with control cells. Moreover, the expression of two known downstream target genes of HIF1α was upregulated. The authors then used three different prediction algorithms alongside previously published data to identify additional miRNA-regulated host genes that could alter glucose metabolism. The genes identified included those encoding the HIF prolyl hydroxylase EGLN2 and the mitochondrial heat shock protein HSPA9. The expression levels of both EGLN2 and HSPA9 were reduced in miRNA-expressing LECs compared with control cells. To probe the connection with the Warburg effect, the authors inhibited the expression of EGLN2 and HSPA9, and found that this stabilized HIF1α and reduced O2 consumption and mitochondrial volume. Notably however, overexpression of either EGLN2 or HSPA9 could not fully reverse the miRNA phenotype, suggesting that other genes are also involved.
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