KSHV episomes: rugged individualists on the factory floor: DOI: 10.14800/rd.1565

Abstract

We have recently developed tools to study Kaposi’s sarcoma-associated virus (KSHV) reactivation at the single-episome level. Using immunofluorescent labeling of latent nuclear antigen (LANA) protein to localize viral episomes, combined with fluorescence in situ RNA hybridization (RNA-FISH) of an intron region of immediate early transcripts, we have visualized active transcription of viral genomes in infected cells. At this level, we observed that not all episomes within a single cell were uniformly transcribed following reactivation stimuli. However, those episomes that were transcribed, formed large aggregates containing a significant fraction of cellular RNA polymerase II (RNAPII), foci consistent with previously described viral transcriptional factories. This focal assembly of RNAPII on viral episomes was accompanied by an overall decrease in the pool of cellular RNAPII. Additionally, the viral transcriptional factories localized with replicating viral genomic DNAs. This co-localization suggests that KSHV may assemble an “all-in-one” workroom for both gene transcription and DNA replication. While previous studies have reported on the variable response of individual KSHV infected cells or episomes derived from a population during reactivation, our results expose this variation further by demonstrating heterogeneity in the response of individual KSHV episomes within a single reactivating cell