Multiplex Detection and Quantitation of Latent and Lytic Transcripts of Human Herpesvirus-8 Using RNase Protection Assay

Human herpesvirus-8 (HHV-8, also called Kaposi's sarcoma-associated herpesvirus) infectious cycle can be divided into latent and lytic phases. During the latent phase viral gene expression is reduced to a minimum, while during the lytic phase, numerous genes are expressed sequentially. The development of an RNase Protection Assay (RPA) is described that allows the detection and quantitation of three latent (ORFs 71-72-73) and three lytic (ORFs 74-K4-K2) HHV-8 genes as well as two cellular housekeeping gene transcripts (glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin) for normalization purposes is described. The RPA was validated using 293T cells transfected with corresponding HHV-8 expression vectors and using resting and phorbol ester-butyric acid-activated BC-3 and BCBL-1 cells. The results obtained indicate that this RPA is specific, sensitive and allows for the simultaneous monitoring of HHV-8 latent and lytic genes expression. HHV-8-RPA is therefore a useful technique to monitor the status of HHV-8 infection in infected cells (latent versus lytic) by comparing and quantitating multiple viral transcripts expression from a single RNA sample.