Ayman El-Guindy

Lytic Cycle Switches of Oncogenic Human Gammaherpesviruses1

The seminal experiments of George and Eva Klein helped to define the two life cycles of Epstein-Barr Virus (EBV), namely latency and lytic or productive infection. Their laboratories described latent nuclear antigens expressed during latency and discovered several chemicals that activated the viral lytic cycle. The mechanism of the switch between latency and the lytic cycle of EBV and Kaposis sarcoma-associated herpesvirus (KSHV) can be studied in cultured B cell lines. Lytic cycle activation of EBV is controlled by two viral transcription factors, ZEBRA and Rta. The homologue of Rta encoded in ORF50 is the lytic cycle activator of KSHV. Control of the lytic cycle can be divided into two distinct phases. Upstream events control expression of the virally encoded lytic cycle activator genes. Downstream events represent tasks carried out by the viral proteins in driving expression of lytic cycle genes and lytic viral DNA replication. In this chapter, we report three recent groups of experiments relating to upstream and downstream events. Azacytidine (AzaC) is a DNA methyltransferase inhibitor whose lytic cycle activation capacity was discovered by G. Klein and coworkers. We find that AzaC rapidly activates the EBV lytic cycle but does not detectably alter DNA methylation or histone acetylation on the promoters of the EBV lytic cycle activator genes. AzaC probably acts via a novel, yet to be elucidated, mechanism. The lytic cycle of both EBV and KSHV can be activated by sodium butyrate (NaB), a histone deacetylase inhibitor whose activity in disrupting latency was also discovered by G. Klein and coworkers. Activation of EBV by NaB requires protein synthesis; activation of KSHV is independent of protein synthesis. Thus, NaB works by a different pathway on the two closely related viruses. ZEBRA, the major downstream mediator of EBV lytic cycle activation is both a transcription activator and an essential replication protein. We show that phosphorylation of ZEBRA at its casein kinase 2 (CK2) site separates these two functions. Phosphorylation by CK2 is required for ZEBRA to activate lytic replication but not to induce expression of early lytic cycle genes. We discuss a number of unsolved mysteries about lytic cycle activation which should provide fertile territory for future research.

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 Epstein-Barr 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 (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by n-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.

Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta.

ABSTRACT ZEBRA, a member of the bZIP family, serves as a master switch between latent and lytic cycle Epstein-Barr virus (EBV) gene expression. ZEBRA influences the activity of another viral transactivator, Rta, in a gene-specific manner. Some early lytic cycle genes, such as BMRF1, are activated in synergy by ZEBRA and Rta. However, ZEBRA suppresses Rtas ability to activate a late gene, BLRF2. Here we show that this repressive activity is dependent on the phosphorylation state of ZEBRA. We find that two residues of ZEBRA, S167 and S173, that are phosphorylated by casein kinase 2 (CK2) in vitro are also phosphorylated in vivo. Inhibition of ZEBRA phosphorylation at the CK2 substrate motif, either by serine-to-alanine substitutions or by use of a specific inhibitor of CK2, abolished ZEBRAs capacity to repress Rta activation of the BLRF2 gene, but did not alter its ability to initiate the lytic cycle or to synergize with Rta in activation of the BMRF1 early-lytic-cycle gene. These studies illustrate how the phosphorylation state of a transcriptional activator can modulate its behavior as an activator or repressor of gene expression. Phosphorylation of ZEBRA at its CK2 sites is likely to play an essential role in proper temporal control of the EBV lytic life cycle.

Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors

ABSTRACT A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory- and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.

Disruption of Epstein-Barr Virus Latency in the Absence of Phosphorylation of ZEBRA by Protein Kinase C

ABSTRACT ZEBRA protein converts Epstein-Barr virus (EBV) infection from the latent to the lytic state. The ability of ZEBRA to activate this switch is strictly dependent on the presence of serine or threonine at residue 186 of the protein (A. Francis, T. Ragoczy, L. Gradoville, A. El-Guindy, and G. Miller, J. Virol. 72:4543-4551, 1999). We investigated whether phosphorylation of ZEBRA protein at this site by a serine-threonine protein kinase was required for activation of an early lytic cycle viral gene, BMRF1, as a marker of disruption of latency. Previous studies suggested that phosphorylation of ZEBRA at S186 by protein kinase C (PKC) activated the protein (M. Baumann, H. Mischak, S. Dammeier, W. Kolch, O. Gires, D. Pich, R. Zeidler, H. J. Delecluse, and W. Hammerschmidt, J. Virol 72:8105-8114, 1998). Two residues of ZEBRA, T159 and S186, which fit the consensus for phosphorylation by PKC, were phosphorylated in vitro by this enzyme. Several isoforms of PKC (α, β1, β2, γ, δ, and ε) phosphorylated ZEBRA. All isoforms that phosphorylated ZEBRA in vitro were blocked by bisindolylmaleimide I, a specific inhibitor of PKC. Studies in cell culture showed that phosphorylation of T159 was not required for disruption of latency in vivo, since the T159A mutant was fully functional. Moreover, the PKC inhibitor did not block the ability of ZEBRA expressed from a transfected plasmid to activate the BMRF1 downstream gene. Of greatest importance, in vivo labeling with [32P]orthophosphate showed that the tryptic phosphopeptide maps of wild-type ZEBRA, Z(S186A), and the double mutant Z(T159A/S186A) were identical. Although ZEBRA is a potential target for PKC, in the absence of PKC agonists, ZEBRA is not constitutively phosphorylated in vivo by PKC at T159 or S186. Phosphorylation of ZEBRA by PKC is not essential for the protein to disrupt EBV latency.

Type I Interferon Regulates the Placental Inflammatory Response to Bacteria and is Targeted by Virus: Mechanism of Polymicrobial Infection-Induced Preterm Birth

Preterm birth (PTB) affects approximately 12% of pregnancies and at least 50% of cases have no known risk factors. We hypothesize that subclinical viral infections of the placenta are a factor sensitizing women to intrauterine bacterial infection. Specifically, we propose that viral‐induced placental IFN‐β inhibition results in a robust inflammatory response to low concentrations of bacteria.

N-linked Glycosylation Is Required for Optimal Function of Kaposi's Sarcoma Herpesvirus-encoded, but Not Cellular, Interleukin 6

Kaposis sarcoma–associated herpesvirus interleukin-6 (vIL-6) is a structural and functional homologue of the human cytokine IL-6 (hIL-6). hIL-6 and vIL-6 exhibit similar biological functions and both act via the gp130 receptor subunit to activate the Janus tyrosine kinase (JAK)1 and signal transducer and activator of transcription (STAT)1/3 pathway. Here we show that vIL-6 is N-linked glycosylated at N78 and N89 and demonstrate that N-linked glycosylation at site N89 of vIL-6 markedly enhances binding to gp130, signaling through the JAK1-STAT1/3 pathway and functions in a cytokine-dependent cell proliferation bioassay. Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor α–dependent binding to gp130 or signaling through JAK1-STAT1/3. As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation. These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6.

Identification of Constitutive Phosphorylation Sites on the Epstein-Barr Virus ZEBRA Protein

ZEBRA, the product of the Epstein-Barr virus gene bzlf1, and a member of the AP-1 subfamily of basic zipper (bZIP) transcription factors, is necessary and sufficient to disrupt viral latency and to initiate the viral lytic cycle. Two serine residues of ZEBRA, Ser167 and Ser173, are substrates for casein kinase 2 (CK2) and are constitutively phosphorylated in vivo. Phosphorylation of ZEBRA at its CK2 sites is required for proper temporal regulation of viral gene expression. Phosphopeptide analysis indicated that ZEBRA contains additional constitutive phosphorylation sites. Here we employed a co-migration strategy to map these sites in vivo. The cornerstone of this strategy was to correlate the migration of 32P- and 35S-labeled tryptic peptides of ZEBRA. The identity of the peptides was revealed by mutagenesis of methionine and cysteine residues present in each peptide. Phosphorylation sites within the peptide were identified by mutagenesis of serines and threonines. ZEBRA was shown to be phosphorylated at serine and threonine residues, but not tyrosine. Two previously unrecognized phosphorylation sites of ZEBRA were identified in the NH2-terminal region of the transactivation domain: a cluster of weak phosphorylation sites at Ser6, Thr7, and Ser8 and a strong phosphorylation site at Thr14. Thr14 was embedded in a MAP kinase consensus sequence and could be phosphorylated in vitro by JNK, despite the absence of a canonical JNK docking site. Thus ZEBRA is now known to be constitutively phosphorylated at three distinct sites.

Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

ABSTRACT The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

ABSTRACT Two transcription factors, ZEBRA and Rta, switch Epstein-Barr virus (EBV) from the latent to the lytic state. While ZEBRA also plays an obligatory role as an activator of replication, it is not known whether Rta is directly required for replication. Rta is dispensable for amplification of an oriLyt-containing plasmid in a transient-replication assay. Here, we assessed the requirement for Rta in activation of viral DNA synthesis from the endogenous viral genome, a function that has not been established. Initially, we searched for a ZEBRA mutant that supports viral replication but not transcription. We found that Z(S186A), a mutant of ZEBRA unable to activate transcription of Rta or viral genes encoding replication proteins, is competent to bind to oriLyt and to function as an origin recognition protein. Ectopic expression of the six components of the EBV lytic replication machinery failed to rescue replication by Z(S186A). However, addition of Rta to Z(S186A) and the mixture of replication factors activated viral replication and late gene expression. Deletion mutagenesis of Rta indicated that the C-terminal 10 amino acids (aa) were essential for the function of Rta in replication. In vivo DNA binding studies revealed that Rta interacted with the enhancer region of oriLyt. In addition, expression of Rta and Z(S186A) together, but not individually, activated synthesis of the BHLF1 transcript, a lytic transcript required for the process of viral DNA replication. Our findings demonstrate that Rta plays an indispensable role in the process of lytic DNA replication.