Lee Heston

Antibodies to Butyrate-Inducible Antigens of Kaposi's Sarcoma–Associated Herpesvirus in Patients with HIV-1 Infection

BACKGROUND The recent identification in patients with Kaposis sarcoma of DNA sequences with homology to gammaherpesviruses has led to the hypothesis that a newly identified virus, Kaposis sarcoma-associated herpeslike virus (KSHV), has a role in the pathogenesis of Kaposis sarcoma. We developed serologic markers for KSHV infection. METHODS KSHV antigens were prepared from a cell line (BC-1) that contains the genomes of both KSHV and the Epstein-Barr virus (EBV). We used immunoblot and immunofluorescence assays to examine serum samples from 102 patients with human immunodeficiency virus type 1 (HIV-1) infection for antibodies to KSHV-associated proteins and to distinguish these antibodies from antibodies to EBV antigens. A positive serologic response was defined by the recognition of an antigenic polypeptide, p40, in n-butyrate-treated BC-1 cells and by the absence of p40 recognition in untreated BC-1 cells or EBV-infected, KSHV-negative cells. The detection by the immunofluorescence assay of 10 to 20 times more antigen-positive cells in n-butyrate-treated BC-1 cells than in untreated cells was considered a positive response. RESULTS Antibodies to the p40 antigen expressed by chemically treated BC-1 cells were identified in 32 of 48 HIV-1-infected patients with Kaposis sarcoma (67 percent), as compared with only 7 of 54 HIV-1-infected patients without Kaposis sarcoma (13 percent). These results were confirmed by an immunofluorescence assay. The positive predictive value of the serologic tests for Kaposis sarcoma was 82 percent, and the negative predictive value 75 percent. CONCLUSIONS The presence of antibodies to a KSHV antigenic peptide correlates with the presence of Kaposis sarcoma in a high-risk population and provides further evidence of an etiologic role for KSHV.

Serologic association of human herpesvirus eight with posttransplant Kaposi's sarcoma in Saudi Arabia

BACKGROUND This study investigates the association between human herpesvirus eight (HHV8) and Kaposis sarcoma (KS), the most common cancer occurring in renal transplant recipients in Saudi Arabia. METHODS A cross-sectional study of seroreactivity to HHV8 antigens in posttransplant KS patients from a tertiary care hospital in Riyadh, Saudi Arabia, and in control subjects without KS was conducted. Seroreactivity rates were determined using immunoblotting assays to detect antibodies to two lytic cycle HHV8 antigens: p40, an antigen found in infected cells, and sVCA, an HHV8-encoded small viral capsid antigen expressed in Escherichia coli. RESULTS Antibodies to HHV8 p40 and sVCA were present in a significantly higher proportion of renal transplant patients with KS (13 of 14 patients) compared to renal transplant patients without KS (5 of 18; P<0.001) and compared to other control individuals (6 of 44; P<0.001). HHV8 seroreactivity was more common among patients with renal failure (28%) than among other control groups (7%). CONCLUSIONS The serologic results provide evidence of a strong association between HHV8 and posttransplant KS in Saudi Arabia.

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.

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.

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

ABSTRACT Epstein-Barr virus (EBV) can be reactivated from latency into the lytic cycle by many stimuli believed to operate by different mechanisms. Cell lines containing EBV differ in their responses to inducing stimuli, yet all stimuli require de novo protein synthesis (44). A crucial step preliminary to identifying these proteins and determining when they are required is to measure the duration of stimulus and response time needed for activation of expression of EBV BRLF1 and BZLF1, the earliest viral indicators of reactivation. Here we show, with four EBV-containing cell lines that respond to different inducing agents, that stimuli that are effective at reactivating EBV can be divided into two main groups. The histone deacetylase inhibitors sodium butyrate and trichostatin A require a relatively long period of exposure, from 2 to 4 h or longer. Phorbol esters, anti-immunoglobulin G (anti-IgG), and, surprisingly, 5-aza-2′-deoxycytidine require short exposures of 15 min or less. The cell/virus background influences the response time. Expression of the EBV BZLF1 and BRLF1 genes can be detected before 2 h in Akata cells treated with anti-IgG, but both long- and short-duration stimuli required 4 or more hr to activate BZLF1 and BRLF1 expression in HH514-16, Raji, or B95-8 cells. Thus, stimulus duration and response time are independent variables. Neither stimulus duration nor response time can be predicted by the number of cells activated into the lytic cycle. These experiments shed new light on the earliest events leading to lytic cycle reactivation of EBV.

Phosphoacceptor Site S173 in the Regulatory Domain of Epstein-Barr Virus ZEBRA Protein Is Required for Lytic DNA Replication but Not for Activation of Viral Early Genes

ABSTRACT The Epstein-Barr virus ZEBRA protein controls the viral lytic cycle. ZEBRA activates the transcription of viral genes required for replication. ZEBRA also binds to oriLyt and interacts with components of the viral replication machinery. The mechanism that differentiates the roles of ZEBRA in regulation of transcription and initiation of lytic replication is unknown. Here we show that S173, a residue in the regulatory domain, is obligatory for ZEBRA to function as an origin binding protein but is dispensable for its role as a transcriptional activator of early genes. Serine-to-alanine substitution of this residue, which prevents phosphorylation of S173, resulted in a threefold reduction in the DNA binding affinity of ZEBRA for oriLyt, as assessed by chromatin immunoprecipitation. An independent assay based on ZEBRA solubility demonstrated a marked defect in DNA binding by the Z(S173A) mutant. The phenotype of a phosphomimetic mutant, the Z(S173D) mutant, was similar to that of wild-type ZEBRA. Our findings suggest that phosphorylation of S173 promotes viral replication by enhancing ZEBRAs affinity for DNA. The results imply that stronger DNA binding is required for ZEBRA to activate replication than that required to activate transcription.

A Subset of Replication Proteins Enhances Origin Recognition and Lytic Replication by the Epstein-Barr Virus ZEBRA Protein

ZEBRA is a site-specific DNA binding protein that functions as a transcriptional activator and as an origin binding protein. Both activities require that ZEBRA recognizes DNA motifs that are scattered along the viral genome. The mechanism by which ZEBRA discriminates between the origin of lytic replication and promoters of EBV early genes is not well understood. We explored the hypothesis that activation of replication requires stronger association between ZEBRA and DNA than does transcription. A ZEBRA mutant, Z(S173A), at a phosphorylation site and three point mutants in the DNA recognition domain of ZEBRA, namely Z(Y180E), Z(R187K) and Z(K188A), were similarly deficient at activating lytic DNA replication and expression of late gene expression but were competent to activate transcription of viral early lytic genes. These mutants all exhibited reduced capacity to interact with DNA as assessed by EMSA, ChIP and an in vivo biotinylated DNA pull-down assay. Over-expression of three virally encoded replication proteins, namely the primase (BSLF1), the single-stranded DNA-binding protein (BALF2) and the DNA polymerase processivity factor (BMRF1), partially rescued the replication defect in these mutants and enhanced ZEBRAs interaction with oriLyt. The findings demonstrate a functional role of replication proteins in stabilizing the association of ZEBRA with viral DNA. Enhanced binding of ZEBRA to oriLyt is crucial for lytic viral DNA replication.

Mutations of amino acids in the DNA-recognition domain of Epstein-Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

ZEBRA, a transcription factor and DNA replication protein encoded by the Epstein-Barr virus (EBV) BZLF1 gene, plays indispensable roles in the EBV lytic cycle. We recently described the phenotypes of 46 single amino acid substitutions introduced into the DNA-recognition region of ZEBRA [Heston, L., El-Guindy, A., Countryman, J., Dela Cruz, C., Delecluse, H.J., and Miller, G. 2006]. The 27 DNA-binding-proficient mutants exhibited distinct defects in their ability to activate expression of the kinetic classes of viral genes. Four phenotypic variants could be discerned: wild-type, defective at activating Rta, defective at activating early genes, and defective at activating late genes. Here we analyze the distribution of ZEBRA within the nucleus and the localization of EA-D (the viral DNA polymerase processivity factor), an indicator of the development of replication compartments, in representatives of each phenotypic group. Plasmids encoding wild-type (WT) and mutant ZEBRA were transfected into 293 cells containing EBV-bacmids. WT ZEBRA protein was diffusely and smoothly distributed throughout the nucleus, sparing nucleoli, and partially recruited to globular replication compartments. EA-D induced by WT ZEBRA was present diffusely in some cells and concentrated in globular replication compartments in other cells. The distribution of ZEBRA and EA-D proteins was identical to WT following transfection of K188R, a mutant with a conservative change. The distribution of S186A mutant ZEBRA protein, defective for activation of Rta and EA-D, was identical to WT, except that the mutant ZEBRA was never found in globular compartments. Co-expression of Rta with S186A mutant rescued diffuse EA-D but not globular replication compartments. The most striking observation was that several mutant ZEBRA proteins defective in activating EA-D (R179A, K181A and A185V) and defective in activating lytic viral DNA replication and late genes (Y180E and K188A) were localized to numerous punctate foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication.

Prospective study of Epstein-Barr virusinfections in acute lymphoblastic leukemia of childhood

A prospective study of Epstein-Barr virus (EBV) infections in children with acutelymphoblastic leukemia (ALL) was undertaken to determine whether EBV is etiologically related to ALL. Five of 12 untreated patients had no serologic evidence for EBV infection at the onset or during the course of leukemia. One infant with congenital leukemia showed a progressive decline in EBV antibody titer to undetectable levels, suggestive of loss of passively acquired maternal antibodies. Six other patients had EBV antibodies at onset, and the titers did not change during follow-up. Lymphoblastoid cell lines were established from three of the seven EBV antibody-positive children and from none of the EBV antibody-negative patients. In addition, two patients with ALL in remission induced by chemotherapy developed infectious mononucleosis. EBV antibodies appeared in their sera and EBV-containing lymphoblastoid cell lines were cultured from them. The results of this study suggest that EBV is not etiologically related to ALL.

Latency of Epstein–Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA

ZEBReplication Activator (ZEBRA), a viral basic zipper protein that initiates the Epstein–Barr viral lytic cycle, binds to DNA and activates transcription through heptamer ZEBRA response elements (ZREs) related to AP-1 sites. A component of the biologic action of ZEBRA is attributable to binding methylated CpGs in ZREs present in the promoters of viral lytic cycle genes. Residue S186 of ZEBRA, Z(S186), which is absolutely required for disruption of latency, participates in the recognition of methylated DNA. We find that mutant cellular AP-1 proteins, Jun(A266S) and Fos(A151S), with alanine-to-serine substitutions homologous to Z(S186), exhibit altered DNA-binding affinity and preferentially bind methylated ZREs. These mutant AP-1 proteins acquire functions of ZEBRA; they activate expression of many viral early lytic cycle gene transcripts in cells harboring latent EBV but are selectively defective in activating expression of some viral proteins and are unable to promote viral DNA replication. Transcriptional activation by mutant c-Jun and c-Fos that have acquired the capacity to bind methylated CpG challenges the paradigm that DNA methylation represses gene expression.