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PubMed1997

The Epstein-Barr virus (EBV) DNA polymerase accessory protein, BMRF1, activates the essential downstream component of the EBV oriLyt.

The EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the "downstream component"), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various protions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from -588 to -592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF-1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication.

Antigens, Viral↗
PubMed1997

The structure and coding organization of the genomic termini of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a novel human herpesvirus closely linked to two AIDS-related neoplasms. We have prepared DNA from KSHV virions produced in cell culture and have examined the structure of the viral genomic termini. As in the related simian herpesvirus H. saimiri (HVS), the central unique region of KSHV DNA is bounded by tandemly repeated units of noncoding, GC-rich DNA. The KSHV repeats are 803 bp in length and are 85% G+C. Each molecule harbors 35-45 such repeats, but the repeats are not arrayed uniformly and symmetrically at each end. Rather, different molecules appear to contain different numbers of repeats at each end, with the sum total of repeated DNA per genome being relatively fixed, since the full genome is uniformly 165-170 kb. Thus, molecules with many repeats at one end will have fewer at the other. Because the unique viral genes bordering the left-hand repeats of the HVS genome play key roles in oncogenesis, we also examined the coding organization of the corresponding region of KSHV. No homologs of the HVS transforming genes were identified in this region of KSHV; rather, this region bears a novel gene encoding a putative transmembrane protein that appears to be upregulated during the early phase of lytic viral replication.

Amino Acid Sequence↗
PubMed2001

Differences in DNA binding specificity among Roseolovirus origin binding proteins.

The Roseolovirus genus of the Betaherpesvirinae consists of the very closely related viruses, human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) plus the somewhat more distantly related human herpesvirus 7 (HHV-7). The roseoloviruses each encode a homolog of the alphaherpesvirus origin binding protein (OBP) which is required for lytic DNA replication. In contrast, members of the other betaherpesvirus genera, the cytomegaloviruses, initiate DNA replication by a different mechanism. To better understand the basis of roseolovirus OBP sequence specificity, we investigated their ability to recognize each other's binding sites. HHV-6A OBP (OBP(H6A)) and HHV-6B OBP (OBP(H6B)) each bind to both of the HHV-7 OBP sites (OBP-1 and OBP-2) with similar strengths, which are also similar to their nearly equivalent interactions with their own sites. In contrast, HHV-7 OBP (OBP(H7)) had a gradient of binding preferences: HHV-7 OBP-2 > HHV-6 OBP-2 > HHV-7 OBP-1 > HHV-6 OBP-1. Thus, the roseolovirus OBPs are not equally reciprocal in their recognition of each other's OBP sites, suggesting that the sequence requirements for the interaction of OBPH7 at the OBP sites in its cognate oriLyt differ from those of OBPH6A and OBPH6B.

Amino Acid Sequence↗
PubMedDate not supplied

In vitro culture of B-lymphocytes derived from Epstein-Barr-virus-associated posttransplant lymphoproliferative disease: cytokine production and effect of interferon-alpha.

Epstein-Barr-virus-associated posttransplant lymphoproliferative disease ranges from transient lymphadenitis to aggressive lymphoma. This study characterizes an in vitro model to study the pathogenesis of this disease with a cell culture system. Five B-cell lines derived from posttransplant lymphoproliferative disease tissue were characterized with regard to immunophenotype, karyotype, molecular genetics, cytokine production, and growth regulation. All cell lines expressed CD19, CD21, CD22, CD43, and CD77, but not CD10 antigens. Immunoglobulin light chain restriction was seen in four of five cell lines, and cytogenetic abnormalities were demonstrable in three of the five. Cells proliferating in culture contained multiple Epstein-Barr virus episomes and showed lytic viral replication. All cell lines produced tumor necrosis factor-beta and interleukin-10 without evidence of autocrine growth regulatory loops involving these cytokines. No evidence of IL-1 alpha, IL-2, IL-4, IL-5 or IL-6 production was found by reverse transcriptase polymerase chain reaction. Adding 500 U IFN-alpha/ml to the culture medium resulted in 30% inhibition of [3H]thymidine incorporation.

Adult↗
PubMed2001

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Bacterial Proteins↗
PubMed2004

Peculiarities of herpes simplex virus (HSV) transcription: an overview.

The herpes simplex virus (HSV) has a 152 kbp dsDNA encoding probably 84 proteins. The approximate number of ORFs is 94, from which seven are doubled. The most probable number of single copy ORFs is 84 after omitting the two latency associated transcripts (LAT)/ORFs and the putative UL27.5 ORF. The high gene number creates a "crowded" genome with several overlapping transcripts. The unique long (U(L)) segment has at least 10 interposed ORFs, the existence of which was not obvious at first sequence analysis, while the unique short (U(S)) segment has two such genes. The surplus of ORFs causes complex transcription patterns: (1) Transcripts with common initiation signals but different termination; (2) Transcripts with different initiation sites but co-terminal ends; (3) "Nested" transcripts differing at both, the initiation as well as termination signals, having partially collinear sequences. At least three or possibly four ORF (gene) pairs (UL9.5/UL10; UL27/UL27.5; UL43/UL43.5; ICP34.5/ORF P and O) occupy both DNA strands at complementary positions rising anti-sense transcripts expressed by an antagonistic mechanism of mutual exclusion. The anti-sense mRNA mechanism might also operate when either LAT or ICP0 ORFs are expressed during latency assuring the absence of lytic virus replication. In contrast, during productive replication the cascade regulation of gene expression predominates, based on stepwise activation of immediate early (IE), early (E), early late (EL) and late (L) promoters. The promoters of different expression kinetic classes (alpha, beta, gamma-1 and gamma-2) are equipped with different number of cellular transcription factor binding and/or enhancer motifs. Surprisingly, only a few HSV mRNAs are being spliced (ICP0, UL15, US1, US12/ICP47). As reviewed here, the transcription pattern of the great majority of overlapping ORFs within the HSV-1 was quite convincingly elucidated, with exception of the putative UL27.5 gene. The UL27.5 transcript was not identified yet. Since the existence of the UL27.5 gene was based on indirect rather than direct evidence, it needs final confirmation.

Base Sequence↗
PubMed1997

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Marek's disease virus: from miasma to model.

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.

Animals↗
PubMed2004

Inhibition of Epstein-Barr virus-induced growth proliferation by a nuclear antigen EBNA2-TAT peptide.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. Antiviral drugs targeted against lytic viral replication have limited efficacy in these disease settings. EBV infection of peripheral blood mononuclear cells induces growth proliferation and the EBV latency Epstein-Barr virus-encoded nuclear antigen (EBNA)2 transcriptional transactivator (TAT) is essential for this response. EBNA2 targets the cellular DNA-binding protein CBF1 to mimic activated Notch signaling. A 10-aa peptide from the CBF1 interaction domain of EBNA2 was synthesized as a fusion with the protein transduction domain of HIV-1 TAT. The EBNA2-TAT peptide blocked EBNA2-CBF1 interaction in an in vitro GST affinity assay and labeling with fluorescein confirmed that the EBNA2-TAT peptide efficiently entered cultured B cells. Neither EBNA2-TAT, nor a mutant peptide with a 2-aa substitution that was unable to block the EBNA2-CBF1 interaction, significantly affected the growth of non-EBNA2-expressing EBV(-) B cells or Burkitt's lymphoma Akata cells. However, treatment of an EBV-immortalized lymphoblastoid cell line with the EBNA2-TAT peptide stopped cell growth and reduced cell viability. RT-PCR analyses of gene expression in the peptide-treated lymphoblastoid cell line cultures revealed that EBNA2-TAT treatment down-regulated the EBNA2-responsive viral LMP1 and LMP2 genes and cellular CD23, intercellular adhesion molecule 1, BATF, and Cdk1 genes while up-regulating expression of the cyclin-dependent kinase inhibitor p21. EBV-induced outgrowth of B cells from cultured peripheral blood mononuclear cells was also blocked in a dose-responsive manner by the EBNA2-TAT peptide. This study suggests that cell-permeable EBNA2 peptides may have potential as novel anti-EBV therapeutics.

Base Sequence↗
PubMed2004

Varicella-zoster virus infection of human neural cells in vivo.

Varicella-zoster virus (VZV) establishes latency in sensory ganglia and causes herpes zoster upon reactivation. These investigations in a nonobese diabetic severe combined immunodeficient mouse-human neural cell model showed that VZV infected both neurons and glial cells and spread efficiently from cell to cell in vivo. Neural cell morphology and protein synthesis were preserved, in contrast to destruction of epithelial cells by VZV. Expression of VZV genes in neural cells was characterized by nuclear retention of the major viral transactivating protein and a block in synthesis of the predominant envelope glycoprotein. The attenuated VZV vaccine strain retained infectivity for neurons and glial cells in vivo. VZV gene expression in differentiated human neural cells in vivo differs from neural infection by herpes simplex virus, which is characterized by latency-associated transcripts, and from lytic VZV replication in skin. The chimeric nonobese diabetic severe combined immunodeficient mouse model may be useful for investigating other neurotropic human viruses.

Animals↗
PubMed1994

An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking.

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.

Amino Acid Sequence↗
PubMed2005

Human cytomegalovirus UL84 is a phosphoprotein that exhibits UTPase activity and is a putative member of the DExD/H box family of proteins.

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 degrees C. The enzyme preferred Mg2+ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn2+, Ca2+, and Zn2+ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.

Amino Acid Sequence↗
PubMed2001

A secreted chemokine binding protein encoded by murine gammaherpesvirus-68 is necessary for the establishment of a normal latent load.

Herpesviruses encode a variety of proteins with the potential to disrupt chemokine signaling, and hence immune organization. However, little is known of how these might function in vivo. The B cell-tropic murine gammaherpesvirus-68 (MHV-68) is related to the Kaposi's sarcoma-associated herpesvirus (KSHV), but whereas KSHV expresses small chemokine homologues, MHV-68 encodes a broad spectrum chemokine binding protein (M3). Here we have analyzed the effect on viral pathogenesis of a targeted disruption of the M3 gene. After intranasal infection, an M3 deficiency had surprisingly little effect on lytic cycle replication in the respiratory tract or the initial spread of virus to lymphoid tissues. However, the amplification of latently infected B cells in the spleen that normally drives MHV-68-induced infectious mononucleosis failed to occur. Thus, there was a marked reduction in latent virus recoverable by in vitro reactivation, latency-associated viral tRNA transcripts detectable by in situ hybridization, total viral DNA load, and virus-driven B cell activation. In vivo CD8(+) T cell depletion largely reversed this deficiency, suggesting that the chemokine neutralization afforded by M3 may function to block effective CD8(+) T cell recruitment into lymphoid tissue during the expansion of latently infected B cell numbers. In the absence of M3, MHV-68 was unable to establish a normal latent load.

Animals↗
PubMed1999

Molecular polymorphism of Kaposi's sarcoma-associated herpesvirus (Human herpesvirus 8) latent nuclear antigen: evidence for a large repertoire of viral genotypes and dual infection with different viral genotypes.

Molecular polymorphism was found in Kaposi's sarcoma-associated herpesvirus (KSHV) latent nuclear antigen (LNA), mapped to the internal repeat domain of the encoding orf73 gene, and used to develop a novel genotyping technique, KSHV LNA genotyping (KVNAtyping). KVNAtype was stable during latent and lytic viral replication in cell culture and in humans. Diverse KVNAtypes were identified in 43 specimens: 6 KSHV cell lines and 6 Kaposi's sarcoma (KS) and 4 primary effusion lymphoma (PEL) tumor samples from the United States, 15 KS tumor samples from Italy, and 12 KS tumor samples from Zambia. A single KVNAtype was detected in each of 41 specimens, and 2 KVNAtypes were detected in each of 2 KS specimens. Multifocal KS from 3 patients showed the same single KVNAtype at all sites in each patient. These results demonstrate a large repertoire of KSHV genotypes and suggest that the development of most KSs and PELs is associated with a single viral genotype.

Antigens, Viral↗
PubMed1999

Identification of transactivator and nuclear localization domains in the Epstein-Barr virus DNA polymerase accessory protein, BMRF1.

The Epstein-Barr virus (EBV) BMRF1 gene product is an essential component of the viral DNA polymerase and is absolutely required for lytic virus replication. In addition to its polymerase accessory protein function, we recently demonstrated that BMRF1 is a transactivator, inducing expression of the essential oriLyt promoter, BHLF1. However, the regions of BMRF1 required for transactivation of BHLF1 are unknown. Here we demonstrate that the carboxy-terminal portion of the BMRF1 protein (amino acids 378404), although not required for DNA binding or polymerase processivity function, is required for transactivator function as well as nuclear localization. Site-directed mutagenesis of this region allowed us to separate the transactivator and nuclear localization motifs of BMRF1. The two DNA-binding domains of BMRF1 are also required for efficient transactivation of the BHLF1 promoter.

Amino Acid Sequence↗
PubMed1974

Relationship between lysogeny, spontaneous induction, and transformation efficiencies in Bacillus subtilis.

The low transformation efficiency of Bacillus subtilis 168 lysogenic for phages ø105 or SPO2 is shown to result from the induction of lytic phage replication in competent cells. Lysogenic competent cells have a higher rate of spontaneous prophage induction than noncompetent cells. Mutants of ø105 and SPO2 which form lysogens resistant to spontaneous induction were isolated, and these lysogens exhibited higher transformation levels than those formed by wild-type phage. These results suggest that the physiological state of competence in B. subtilis promotes prophage derepression leading to cell death and the loss of potential transformants.

Bacillus subtilis↗
PubMed2006

Functional characterization of Kaposi's sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis.

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.

Blotting, Western↗
PubMed1989

Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions.

Cells latently infected with Epstein-Barr virus (EBV) can be activated to express lytic-cycle polypeptides by the introduction of the EBV-encoded Z transactivator, indicating that this protein has a pivotal role in virus reactivation. We examined the target specificity of the Z transactivator in short-term contransfection assays and found that the most responsive target to Z transactivation was the divergent NotI repeat promoter, located within the EBV BamHI H fragment. In contrast, target plasmids containing the cat gene linked to heterologous viral promoters were not activated by cotransfection with the Z gene. S1 nuclease analysis of RNA from chemically induced B95-8 cells and from Vero cells cotransfected with NotI repeat promoter-CAT and Z showed that Z transactivation increased the level of correctly initiated, stable RNA transcripts. The NotI repeat gene (ntr) gives rise to a highly abundant mRNA species after chemical induction of lytic virus replication, but no protein product had been previously identified. Using monospecific antiserum raised against a synthetic peptide from the BHLF1 open reading frame, we demonstrated that the ntr gene encodes a protein product that is found in nuclear patches colocalizing with nucleoli. A series of deletions introduced into the upstream sequences of the NotI-repeat-promoter revealed two separate Z-response regions. The minimal promoter region between -7 and -155 of the leftward RNA cap site and an upstream region between -644 and -902 were both independently capable of conferring Z responsiveness. However, the minimal region, which was activated by Z cotransfection in Vero cells, was poorly responsive in lymphocytes, whereas the response of the far-upstream region to Z cotransfection was lymphocyte specific. In its human host, EBV infects both epithelial and lymphocyte populations. This dual lifestyle may have led to the evolution of multiple Z-response signals that enable the Z transactivator to interact with both cell-specific promoter and enhancer factors.

Animals↗