We have discovered and published around 40 validated cellular mRNA targets of KSHV miRNAs. Importantly, many of these targets have not previously been studied in the context of KSHV infection. We found that many of these targets interact in networks that may indicate key host factors that regulate viral infection. We found that many miRNA targets (GRB2, PKCD, MET, BIRC5, IRAK1, EPOR, N4BP2, GADD45B) that we identified, directly interact in a network centered on STAT3. We reported that KSHV miRNAs regulated STAT3 and STAT5 activation upon cytokine treatment and that KSHV miRNAs targeting this same network suppressed the induction of antiviral interferon-stimulated genes upon interferon treatment. Additionally, we investigated translational applications of our discoveries. For example, suppression of STAT3 activity can trigger induction of the lytic cycle, and we demonstrated that STAT3 inhibition in combination with ganciclovir could potentially be used to flush out latent reservoirs of KSHV infection. A transcriptional target of STAT3 is growth arrest and DNA-damage-inducible, beta (GADD45B), which is also repressed by KSHV miRNAs, and we showed that repression of GADD45B protects infected cells from cell cycle arrest and apoptosis. We found that inhibition of one KSHV miRNA that represses GADD45B increased GADD45B expression and apoptosis in latently infected cells. GADD45B is resistant to KSHV-mediated host gene shutoff during the lytic cycle and KSHV miRNAs may provide an additional level of regulation of the GADD45B transcript. Additionally, we found that MCPIP1 expression and 25-hydroxycholesterol (25HC) can inhibit KSHV infections, which could be translated into new antiviral therapies. It should be noted that MCPIP1 and 25HC have also been shown to inhibit HIV infection. Since EBV and KSHV microRNAs are predicted to target a majority of the same human genes, our analysis will likely be relevant in other viral infections. In summary, our work over the last several years identified and validated many miRNA targets and their associated functions (BCR, TPM1, STAT network, GADD45B, and others) in changes to cellular behavior. Understanding the interactions between human miRNA regulators and KSHV miRNAs After the discovery of viral miRNAs, it was assumed that they could not be specifically targeted for degradation by host factors. However, the recent discovery that monocyte chemoattractant protein induced protein (MCPIP1) can cleave human miRNA precursor molecules raised the possibility that the host could inhibit biogenesis of viral miRNAs, especially in the context of inflammatory signals, since MCPIP1 expression is induced by multiple inflammatory signals. We discovered that MCPIP1 can degrade the majority of KSHV miRNA precursors, but also that a specific KSHV miRNA, which is protected from MCPIP1 degradation, can directly target the MCPIP1 transcript and decrease MCPIP1 expression. We discovered that upon KSHV infection, MCPIP1 expression is decreased. MCPIP1 is also repressed by Epstein-Barr Virus (EBV/HHV4) infection and MCPIP1 can also cleave at least two EBV pre-miRNAs. Together, the data suggest that herpesvirus infections can promote biogenesis of their viral miRNAs by inhibiting a host factor that represses miRNA biogenesis and by promoting expression of host factors that promote miRNA biogenesis. We have identified a sequence motif that is enriched in KSHV pre-miRNAs and MCPIP1-targeted human pre-miRNAs. Mutation of this motif disrupts MCPIP1-mediated degradation of pre-miRNAs. Our research on MCPIP1 may lead to new ways to selectively degrade viral miRNAs as new antiviral strategies. Finally, we have found that expression of MCPIP1 inhibits KSHV infection. Understanding the consequences of KSHV miRNA-driven repression of multiple genes in the cholesterol (mevalonate) pathway and how the cholesterol pathway affects viral infection Using proteomic screening, we found and validated HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1) as the host protein that was most repressed by KSHV viral miRNAs. HMGCS1 is an enzyme of the mevalonate pathway, which synthesizes cholesterol, among other important functions. We have observed that intracellular cholesterol levels decrease in the presence of KSHV miRNAs and have also identified several other targets of KSHV viral miRNAs in the mevalonate pathway. We also discovered that 25-hydroxycholesterol (25HC), a product of cholesterol, strongly inhibits KSHV infection and others have recently shown that it can inhibit another viral infection of human primary cells. We are continuing to investigate the effects of KSHV infection on the mevalonate pathway and determining the mechanism of how 25HC inhibits viral infection. Our recent RNA-sequencing data has shown that 25HC triggers a strong innate immune response, including some important genes for an interferon response to KSHV infection. We are investigating translational applications of this information, which could lead to strategies to inhibit multiple viral infections or to improve cancer immunotherapy.
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