Oncogenic gammaherpesviruses express viral products during latent and lytic
infection that block the innate immune response. Previously, we found that
Kaposi's sarcoma herpesvirus (KSHV/human herpesvirus-8) viral
microRNAs (
miRNAs) downregulate
cholesterol biogenesis, and we hypothesized that this prevents the production of
25-hydroxycholesterol (25HC), a
cholesterol derivative. 25HC blocks KSHV de novo
infection of primary endothelial cells at a postentry step and decreases viral gene expression of LANA (
latency-associated nuclear antigen) and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using
RNA sequencing (
RNA-Seq). We found that 25HC treatment inhibited KSHV gene expression and induced
interferon-stimulated genes (ISGs) and several inflammatory
cytokines (
interleukin 8 [IL-8], IL-1α). Some 25HC-induced genes were partially responsible for the broadly
antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited
infection of primary B cells by a related oncogenic virus, Epstein-Barr virus (EBV/human herpesvirus-4) by suppressing key viral genes such as LMP-1 and inducing apoptosis.
RNA-Seq analysis revealed that
IL-1 and
IL-8 pathways were induced by 25HC in both primary endothelial cells and B cells. We also found that the gene encoding
cholesterol 25-hydroxylase (CH25H), which converts
cholesterol to 25HC, can be induced by
type I interferon (IFN) in human B cell-enriched peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral
miRNAs target the
cholesterol pathway to prevent 25HC production and subsequent induction of
antiviral ISGs. Together, these results answer some important questions about a widely acting
antiviral (25HC), with implications for multiple viral and
bacterial infections. IMPORTANCE A
cholesterol derivative,
25-hydroxycholesterol (25HC), has been demonstrated to inhibit
infections from widely different bacteria and viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, its mechanism of activity is still not fully understood. In this work, we look at gene expression changes in the host and virus after 25HC treatment to find clues about its
antiviral activity. We likewise demonstrate that 25HC is also
antiviral against EBV, a common
cancer-causing virus. We compared our results with previous data from
antiviral screening assays and found the same pathways resulting in
antiviral activity. Together, these results bring us closer to understanding how a modified form of
cholesterol works against several viruses.