BZLF1

Abstract: PERMISSIVE cell systems for the replication of the Epstein–Barr virus (EBV) and some of the related EBV-like agents (for example, Herpesvirus papio and herpesvirus pan) have not yet been established1. EBV is regularly demonstrated in a small number of cells from lymphoblastoid lines of B-cell origin which have been derived from certain lymphoma patients, most frequently from Burkitt's lymphoma or patients with infectious mononucleosis, but also from healthy EBV-reactive individuals. EBV DNA usually persists in cells of these lines in multiple copies2–4, but spontaneous induction of viral antigens and particle synthesis occurs in a majority of the lines at a low rate. To some extent, the percentage of induced cells seems to be cell line-specific1, and two lines which are rather high producers of EBV are the P3HR-I line of BL origin5 and the marmoset B95-8 line isolated from lymphocytes transformed with EBV of infectious mononucleosis origin6. At any time during cultivation, 2–10% of the cells reveal viral capsid antigen synthesis as determined by indirect immunofluorescence7. All attempts to increase the virus yield in these lines by temperature shifts or chemical or physical inducers (IUdR, mitomycin C, X rays) have usually resulted in only barely significant enhancement of viral replication8–10. We now report on a very efficient induction of EBV and other oncogenic herpesviruses by the well established cocarcinogen and tumour promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA)11. In addition some TPA-treated cells show cytopathogenic changes, including polyploidisation. These observations may enable a more complete investigation of the virus-cell-gene balance hypothesis12.

Abstract: In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10-, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

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.