Kinetics of Kaposi’s Sarcoma-Associated Herpesvirus Gene Expression

Abstract

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the “immediate-early” gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

KSHV vFLIP Is Essential for the Survival of Infected Lymphoma Cells

“…1), and compared the amount of transcripts in latently infected PEL cells (BC1, BC2, and BC3) to the level of NF-B as measured by EMSAs in these three PEL cell lines. This analysis confirmed previous analyses (27,28) in-dicating that K1, vGPCR, and K15 are lytic genes that are not significantly expressed by latently infected PEL cells, whereas vFLIP is expressed at significant levels during latency. In addition, we found that the transcript encoding vFLIP is present in PEL cells at levels that correlate with the degree of NF-B DNA-binding activity ( Fig.…”

MicroRNAs encoded by Kaposi's sarcoma‐associated herpesvirus regulate viral life cycle

“…As the key components of the miRNA biogenesis machinery are Dicer and Argonaute 2 (AGO2; Chu & Rana, 2007), we knocked down human Dicer and AGO2 using short hairpin RNAs (shRNAs) in latently infected BC-3 cells. These are derived from human primary effusion (body-cavity based) lymphoma, and only a few per cent spontaneously reactivate (Sun et al, 1999).…”

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

“…To control for loading and integrity of the mRNA, the same blots were re-probed with GAPDH ( Figure 2B). Hybridizing the same blots with a probe to a KSHV lytic gene product, PAN (Sun et al, 1996(Sun et al, , 1999, con®rmed successful induction of the viral lytic cycle ( Figure 2B).…”