MbShea@PWP@English@UMCP

Abstract: From various screens, we have found that KSHV viral miRNAs target several enzymes in the mevalonate/cholesterol pathway. HMGCS1 (3-hydroxy- 3-methylglutaryl- CoA synthase1), HMGCR (3-hydroxy- 3-methylglutaryl- CoA reductase, rate-limiting step in the mevalonate pathway), and FDFT1 (farnesyl-diphosphate farnesyltransferase1, committed step in cholesterol branch) are repressed by multiple KSHV miRNAs. Transfecting viral miRNA mimics in primary endothelial cells (HUVEC) is sufficient to reduce intracellular cholesterol levels. However, siRNAs targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly-regulated pathway. We also report here that cholesterol levels were decreased in de novo infected HUVEC cells after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the gene responsible for generating 25HC, CH25H (cholesterol 25-hydroxylase), increased in expression in de novo infected HUVEC, but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC.

Voice: Since this is a lab document that was submitted for a publication, a formal voice is used. Since this research was done by my research group, I will be using first person to portray our group efforts.

Citation: This paper will use formal document citations throughout.

Reader’s Profile: This document is meant for scientists who specialize in AIDS or cancer research, and learning ways to find cures that come from an immunotherapy approach.

Reader's Response: While we did find data that suggests that CH25H and 25HC increase as part of an initial antiviral response to KSHV, The specific mechanisms of how 25HC inhibits KSHV infection remain to be fully understood. Understanding this specific mechanism could open up new possibilities for treatment of KSHV.

O -- looks like a good plan. About voice, how your colleagues handle this choice in research documents? I would expect that most of the document is written in third person? Yet, some strategic use of first person, to signal that the content is now directly related to laboratory work, rather than the broader concerns of the field.