Protein Kinase C-Independent Activation of the Epstein-Barr Virus Lytic Cycle

Abstract

The protein kinase C (PKC) pathway has been considered to be essential for activation of latent EpsteinBarr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase ( The switch between latency and the productive lytic cycle of Epstein-Barr virus (EBV) has been studied extensively in cultured lymphoid cell lines, a system that is advantageous for study of the molecular mechanism of the switch. Viral genes that are expressed during latency rather than during the lytic cycle are well characterized (31). The major viral products that activate the lytic cycle, the transcriptional activators encoded by the genes BZLF1 and BRLF1, have been identified (10,11,26). A number of different stimuli have been shown to trigger the switch into the lytic cycle. These include halogenated pyrimidines (25), nutrient starvation (27), phorbol esters (61), calcium ionophores (16), transforming growth factor ␤1 (17), n-butyrate (36), specific histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) (9, 29), the demethylating agent azacytidine (5), cross-linking of surface immunoglobulin (53), superinfection with EBV stocks containing rearranged defective DNA (41, 43), and superinfection with human herpesvirus 6 (21). A general unanswered question is whether all inducing agents operate through a final common pathway.The mechanisms mediating control of EBV lytic-cycle gene expression are understood in rough outline, but many important details are missing. During latency the genes BRLF1 and BZLF1, encoding the lytic-cycle activators, are repressed. No mRNA or proteins encoded by these genes are detectable during latency (55). Inducing stimuli that activate the lytic cycle lead to expression of BRLF1 and BZLF1.

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

“…For herpesviruses, the ability to induce replication depends on not only the type of CMR but also the type of cell line being induced (Gradoville et al, 2002). The lack of persistently infected cell lines does not help attempts to isolate CFPHV, so we tried to get around this problem by using early subculture cells known to contain CFPHV pol DNA.…”

Measuring T-cell responses against LCV and CMV in cynomolgus macaques using ELISPOT: Potential application to non-clinical testing of immunomodulatory therapeutics

“…It has been reported that latently infected LCL can be driven to enter into the lytic phase of the EBV life cycle by exposure to PMA, a protein kinase C agonist, and sodium butyrate (NaB), a histone deacetylase inhibitor (Bhaduri-McIntosh & Miller, 2006;Gradoville et al, 2002), so protein lysates were prepared from LCL that were either treated with PMA þ NaB or left untreated. In three out of four animals tested, a higher frequency of LCV-specific cells was detected when lysate from PMA/NaBtreated LCL was used as the source of LCV antigen (Figure 2a).…”

“…The presence of LCV genome and protein expression of LCV latent and lytic phase proteins were confirmed (refer to supplemental data section for additional details). Lysates were prepared from non-treated LCL or LCL treated with 20 ng phorbol-12-myristate 13-acetate (PMA)/ml (Sigma, St. Louis, MO) þ 3 mM sodium butyrate (NaB) (Sigma) for 72 h to enhance LCV gene expression (Gradoville et al, 2002). Lot-specific titers for use in the IFN-g ELISPOT assay were determined from dose-response curves; both LCV seronegative and seropositive PBMC were routinely tested to ensure specificity of responses.…”

Virally targeted therapies for EBV-associated malignancies

“…The inducing effect of phorbol esters is mediated through protein kinase C (PKC) (Davies et al, 1991), and also primarily requires the ZI and ZII binding motifs in the BZLF1 promoter , although the intermediary factors are not well defined. However, PKC activation is not required for induction of lytic EBV by certain stimuli (Gradoville et al, 2002). Activation of the BRLF1 (Rp) promoter requires EGR-1 and Sp1 binding sites (Zalani et al, 1992(Zalani et al, , 1995.…”