Stimulus Duration and Response Time Independently Influence the Kinetics of Lytic Cycle Reactivation of Epstein-Barr Virus

Abstract

Epstein-Barr virus (EBVOncogenic human herpesviruses, such as Epstein-Barr virus (EBV), manifest two distinct lifestyles: latency, a state of limited viral gene expression, and lytic replication, which ultimately leads to production of virions. The switch between latency and productive lytic infection can be manipulated in cell culture. Lymphoid cell lines are unique experimental systems with which to study physiologic and molecular mechanisms underlying the transition between the two life cycles. The switch between viral latency and lytic replication is a biologically interesting and potentially tractable example of the combinatorial control of eukaryotic gene expression. Groups of viral and cellular effector molecules, some of which are transcription factors, exert both positive and negative control on the expression and the activity of two virally encoded proteins, ZEBRA and Rta, both of which act as transcription factors and replication proteins. Epigenetic control of viral and cellular gene expression, through chromatinization and DNA methylation, may also play roles in the latency-to-lytic cycle transition. The latency-to-lytic cycle switch has obvious implications for pathogenesis. While latency may be the predominant state of the life cycle in cellular reservoirs and in virus-associated cancers, the viruses must replicate lytically in order to be transmitted between cells and among individuals. Manipulation of the latency-to-lytic cycle switch has been investigated as a potential oncolytic strategy (11,28,36,43).The latency-to-lytic switch can be envisioned to be composed of two distinct complex sets of events: upstream events lead to the expression of the EBV lytic cycle activator genes BZLF1 and BRLF1, which encode ZEBRA and Rta, and downstream events involve the effects of ZEBRA and Rta and their target genes on viral and cellular gene expression, DNA replication, and viral and cellular behavior in general. Upstream events can be initiated in cell culture by the addition of certain inducing stimuli which presumably mimic asyet poorly characterized physiologic stimuli that trigger the latency-to-lytic cycle switch in vivo. A partial list of stimuli that can activate the latency-to-lytic switch in cultured B-cell lines includes phorbol esters (47), which are protein kinase C (PKC) agonists; sodium butyrate (NaB) (26) and trichostatin A (TSA) (46), which are histone deacetylase inhibitors (HDACi); 5-aza-2-Ј-deoxycytidine (AzaCdR) (4), which is a DNA methyltransferase inhibitor; and anti-immunoglobulin G (anti-IgG), which activates the B-cell antigen receptor (40).Inspection of this list of inducing stimuli, which are thought to operate by different modes of action, leads to the conclusion that upstream events are likely to activate different pathways which lead to BZLF1 and BRLF1 expression. These pathways may or may not converge on a final common event. Further complexity in understanding the upstream events is evident from the observation that not all cell/virus systems respond to the same inducing sti...

Cellular Immediate-Early Gene Expression Occurs Kinetically Upstream of Epstein-Barr Virus bzlf1 and brlf1 following Cross-Linking of the B Cell Antigen Receptor in the Akata Burkitt Lymphoma Cell Line

“…Moreover, the response to anti-IgG is very rapid. The mRNAs of bzlf1 and brlf1 can be detected in Akata cells within 2 h after exposure to anti-IgG (11). These findings initially suggested that the pathway of lytic induction by anti-IgG in Akata cells might be qualitatively different from lytic induction by HDAC inhibitors NaB and TSA, 5AzaCdR, and TPA.…”

“…The lytic cycle-inducing agents vary in the duration of exposure required to elicit a response. Moreover, the cells vary in their response time (11). It is not yet known whether the many pathways engaged by the diverse stimuli converge on a final event.…”

“…In subsequent studies we found that the stimuli that reactivate EBV lytic gene expression could be divided into two main groups (11). A relatively long exposure time, from 2 to 4 h or longer, was required for the HDAC inhibitors, NaB and TSA, to reactivate bzlf1 and brlf1 expression (14).…”

“…In Akata cells, anti-IgG acts as a very short duration stimulus. Less than 5 min of exposure to anti-IgG is adequate to induce bzlf1 and brlf1 expression (11). Moreover, the response to anti-IgG is very rapid.…”

Valproic Acid Antagonizes the Capacity of Other Histone Deacetylase Inhibitors To Activate the Epstein-Barr Virus Lytic Cycle

“…We have studied extensively the effects of the HDAC inhibitors NaB and TSA and the DNA methyltransferase inhibitor AzaCdR on viral reactivation (22,24,26,41,64,102). We use the Burkitt lymphoma (BL) cell line HH514-16 for these studies, since these cells have a very low level of spontaneous reactivation and are readily induced by NaB, TSA, or AzaCdR.…”