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At least 163 records · Page 9Linked to original sources
PubMed1993

Analysis of the promoter sequences of the UL9 gene of herpes simplex virus type 1.

The origin-binding protein, encoded by the UL9 gene of herpes simplex virus-1 (HSV-1), has the properties of an initiator of DNA replication. Since onset of lytic replication of the virus can be critically controlled by availability of the initiator protein, we have analyzed the sequences upstream of the UL9 gene in a monkey kidney cell line (Vero). In this communication, we report the presence of three UL9 transcripts and multiple transcription initiation sites of the UL9 gene in HSV-1-infected cells. The presence of multiple transcription initiation sites for the UL9 gene is consistent with the absence of a TATA element 5'- to the UL9 ORF. A 395-bp upstream sequence encompassing all UL9 transcription initiation sites was active in expressing a reporter gene and was induced by viral immediate-early proteins. This result is consistent with the earlier observation that immediate-early proteins of HSV-1 induce viral early genes.

Animals↗
PubMed2003

The role of Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 regulator of transcription activation (RTA) in control of gene expression.

The mechanisms that control the replication state, latency versus lytic, of human herpesviruses have been under intense investigations. Here we summarize some of the recent findings that help define such mechanisms for Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8 (KSHV/HHV-8). For HHV-8, the viral regulator of transcription activation (RTA) is a key mediator of the switch from latency to lytic gene expression in infected cells. RTA is necessary and sufficient to drive HHV-8 lytic replication and the production of viral progeny. The RTA is an immediate-early gene product, it is the initial activator of expression of a multitude of viral and cellular genes that have been implicated in the replication of HHV-8 and pathogenesis of KS. Interactions of RTA with a number of viral promoters, and with a number of transcription factors or transcriptional co-activators are highlighted. Modulation of transactivation, through alternate RTA-protein, or RTA-promoter interactions, is hypothesized to participate in the selective tissue tropism and differential pathogenesis observed in KS.

Gene Expression Regulation, Neoplastic↗
PubMed2002

Ovine herpesvirus 2 lytic cycle replication and capsid production.

Ovine herpesvirus 2 (OvHV-2) causes malignant catarrhal fever in cattle, pigs and deer. We have observed intact circular and linear OvHV-2 genomes in infected T cell lines derived from cows and rabbits. Bovine T cell lines were predominantly latently infected but rabbit T cell lines supported OvHV-2 productive cycle gene expression and virus capsids were demonstrated for the first time.

Animals↗
PubMed1996

The bacteriophage P1 lytic replicon: directionality of replication and cis-acting elements.

We have identified the direction of replication of a bacteriophage P1 lytic replicon. This was accomplished by constructing lambda P1 lysogens that contain a functional P1 lytic replicon and analysing which of two nearby bacterial DNA markers flanking the lambda prophage were amplified when that replicon was activated. We demonstrate that both DNA markers are coordinately amplified, a result consistent with lytic replication proceeding in a bidirectional fashion. To analyze the role of various elements comprising the lytic replicon, we assessed the ability of a wild type replicon to complement a defective replicon that contains a transposon inserted between an essential lytic replication gene (repL) and the promoter (P53) at which transcription of that gene is initiated. We show that the wild type replicon cannot complement the mutant replicon. The simplest hypothesis to explain this result is that either P53 or repL protein functions primarily in cis for the replicon to operate.

Bacteriophage P1↗
PubMed2003

Transcription program of murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68 [also referred to as gammaHV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV). However, unlike KSHV or EBV, MHV-68 readily infects fibroblast and epithelial cell lines derived from several mammalian species, providing a system to study productive and latent infections as well as reactivation of gammaherpesviruses in vivo and in vitro. To carry out rapid genome-wide analysis of MHV-68 gene expression, we made DNA arrays containing nearly all of the known and predicted open reading frames (ORFs) of the virus. RNA obtained from an MHV-68 latently infected cell line, from cells lytically infected with MHV-68 in culture, and from the lung tissue of infected mice was used to probe the MHV-68 arrays. Using a tightly latent B-cell line (S11E), the MHV-68 latent transcription program was quantitatively described. Using BHK-21 cells and infected mice, we demonstrated that latent genes are transcribed during lytic replication and are relatively independent of de novo protein synthesis. We determined that the transcription profiles at the peak of lytic gene expression are similar in cultured fibroblast and in the lung of infected mice. Finally, the MHV-68 DNA arrays were used to examine the gene expression profile of a recombinant virus that overexpresses replication and transcription activator (RTA), C-RTA/MHV-68, during lytic replication in cell culture. The recombinant virus replicates faster then the parental strain and the DNA arrays revealed that nearly every MHV-68 ORF examined was activated by RTA overexpression. Examination of the gene expression patterns of C-RTA/MHV-68 over a time course led to the finding that the M3 promoter is RTA responsive in the absence of other viral factors.

Animals↗
PubMed1994

Negatively cis-acting elements in the distal part of the promoter of Epstein-Barr virus trans-activator gene BZLF1.

Epstein-Barr virus (EBV) replicates in a latent or a lytic way in the infected organism, depending on the type and level of differentiation of the host cell. The switch between latency and lytic replication was previously shown, for Burkitt's lymphoma cell lines, to depend on the viral BZLF1 gene product. Protein-DNA assays were used to identify the cis-acting elements that represent the link between regulating signal transduction pathways and the viral cascade of gene expression. Specific binding of proteins to several sites of the BZLF1 promoter during latency was shown. Induction of the lytic cycle by stimulation with 12-O-tetradecanoyl-phorbol 13-acetate abolished the binding of these proteins to the distal promoter (positions -227 to -551), suggesting a functional role for the down-regulation of promoter activity during latency. Computer analysis identified a multiply repeated sequence motif, HI, in this region and exonuclease III footprints confirmed that these sites act as specific protein recognition sites. Using a set of reporter plasmids we were able to demonstrate a negative regulatory effect of the HI motif in some B lymphoid cell lines, in contrast to epithelial HeLa cells. The HI silencer elements are different from other silencer elements described so far in respect of their sequence and protein-binding pattern during the activation of BZLF1.

Base Sequence↗
PubMed1999

Long-term infection and transformation of dermal microvascular endothelial cells by human herpesvirus 8.

Human herpesvirus 8 (HHV8) infects Kaposi's sarcoma (KS) spindle cells in situ, as well as the lesional endothelial cells considered to be spindle cell precursors. The HHV8 genome contains several oncogenes, suggesting that infection of endothelial and spindle cells could induce cellular transformation and tumorigenesis and promote the formation of KS lesions. To investigate the potential of HHV8 infection of endothelial cells to contribute to the development of KS, we have developed an in vitro model utilizing dermal microvascular endothelial cells that support significant HHV8 infection. In contrast to existing in vitro systems used to study HHV8 pathogenesis, the majority of dermal endothelial cells are infected with HHV8 and the viral genome is maintained indefinitely. Infection is predominantly latent, with a small percentage of cells supporting lytic replication, and latency is responsive to lytic induction stimuli. Infected endothelial cells develop a spindle shape resembling that of KS lesional cells and show characteristics of a transformed phenotype, including loss of contact inhibition and acquisition of anchorage-independent growth. These results describe a relevant model system in which to study virus-host interactions in vitro and demonstrate the ability of HHV8 to induce phenotypic changes in infected endothelial cells that resemble characteristics of KS spindle cells in vivo. Thus, our results are consistent with a direct role for HHV8 in the pathogenesis of KS.

Agar↗
PubMed2005

Early establishment of gamma-herpesvirus latency: implications for immune control.

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, infect >90% of the population worldwide, and latent infection is associated with numerous malignancies. Rational vaccination and therapeutic strategies require an understanding of virus-host interactions during the initial asymptomatic infection. Primary EBV infection is associated with virus replication at epithelial sites and entry into the circulating B lymphocyte pool. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells. In this study, using a murine gamma-herpesvirus model, we demonstrate an early dominance of latent virus at the site of infection, with lung B cells harboring virus almost immediately after infection. These data reinforce the central role of the B cell not only in the later phase of infection, but early in the initial infection. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus. These data demonstrate that lytic viral replication is not a requirement for gamma-herpesvirus latency in vivo and suggest that viral latency can be disseminated by cellular proliferation. These observations emphasize that prophylactic vaccination strategies must target latent gamma-herpesvirus at the site of infection.

Animals↗
PubMed2005

The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor.

Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (DeltaZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.

Animals↗
PubMed2006

Structural basis of lytic cycle activation by the Epstein-Barr virus ZEBRA protein.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.

Amino Acid Sequence↗
PubMed2004

The evolutionarily conserved Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with REF protein and acts as an RNA export factor.

ORF57 (MTA) one of the earliest Kaposi's sarcoma-associated herpesvirus (KSHV) regulatory proteins to be expressed is essential for virus lytic replication. A counterpart is present in every herpesvirus sequenced, indicating the importance of this signature viral protein and those examined act post-transcriptionally, affecting RNA splicing and transport. In KSHV-infected cells, ORF57 protein was present in a complex with REF (Aly) and TAP (NXF1), factors involved in cellular mRNA export. The ORF57 N-terminal region interacts with REF, whereas both N- and C-terminal domains of REF interact with ORF57. The ORF57-REF interaction was direct, whereas TAP appeared to be recruited via REF. In somatic cells, ectopically expressed ORF57 protein was shown to function as a CRM1-independent nuclear mRNA export factor, promoting export of mRNAs that are poor substrates for splicing. The gamma-herpesvirus ORF57 protein, and its alpha-1 herpesvirus ICP27 counterpart both export RNA through pathways involving REF and TAP proteins, although divergence of these herpesvirus subfamilies occurred some 180-210 million years ago. The TAP-mediated cellular mRNA export pathway is CRM1-independent. However, human immunodeficiency virus type 1 Rev protein-mediated RNA export, which is CRM1-dependent, was considerably inhibited by ORF57, suggesting that Rev and ORF57 compete for a common export component. These data strengthen arguments that TAP and CRM1 pathways converge in accessing similar components of the nuclear pore complex. We propose that ORF57-mediated RNA export may use different export factors to accommodate the KSHV-infected host cell environments, for example, in B-cells or endothelial cells and during the different phases of lytic virus replication.

Active Transport, Cell Nucleus↗
PubMed1996

Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication.

During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells. It is not known, however, what the early events in viral DNA replication that yield these concatemers are. In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed. As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA). A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus. This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction. The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids. Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication.

Cell Line↗
PubMed2003

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin.

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.

Base Sequence↗
PubMed2002

Activation of cellular and heterologous promoters by the human herpesvirus 8 replication and transcription activator.

The key regulator of the switch from latent to lytic replication of the human herpesvirus 8 (HHV-8; KSHV) is the replication and transcription activator (Rta). The ability of Rta to regulate cellular gene expression was examined by transient transfection into cells that were not infected with HHV-8. Rta induced some, but not all, NF-kappa B-responsive reporters through mechanisms that did not involve activation of classic forms of NF-kappa B. Furthermore, transfection of the NF-kappa B subunit Rel A inhibited the ability of Rta to transactivate some but not all reporters. For example, Rel A inhibited the ability of Rta to transactivate the IL-6 promoter, but only when sequences upstream of the NF-kappa B site were present. The ability of Rel A to inhibit Rta-mediated transactivation was not dependent on a functional NF-kappa B site within the promoter, suggesting an indirect mechanism for inhibition. These studies suggest that Rta expression during lytic reactivation of HHV-8 would lead to expression of some cellular genes, including IL-6, whereas activation of NF-kappa B could inhibit some responses to Rta.

Binding Sites↗
PubMed1993

A transcription factor with homology to the AP-1 family links RNA transcription and DNA replication in the lytic cycle of Epstein-Barr virus.

oriLyt, the lytic origin of DNA replication of Epstein-Barr virus (EBV), ensures viral DNA amplification during the productive or lytic phase of the virus' life cycle. To understand the contribution of cis- and transacting elements involved in DNA replication of oriLyt, a detailed mutational analysis was undertaken which defined BZLF1, a viral transcriptional activator, as an essential replication factor. The BZLF1 protein belongs to the extended fos/jun family of transcription factors and binds to specific BZLF1 binding motifs within oriLyt, as well as to consensus AP-1 sites. Recombinant, chimeric transcription factors identified the transcriptional activation domain of BZLF1 as being necessary to mediate DNA replication, a function which could not be substituted by any other transcription factor tested, including jun, E2, myc or VP16.

Binding Sites↗
PubMedDate not supplied

Latent and lytic Epstein-Barr virus replication strategies.

The Epstein-Barr virus (EBV) can choose between two alternative lifestyles; latent or lytic replication. In the latent state, the EBV genomic DNA, which exists as a closed circular plasmid, appears to behave just like host chromosomal DNA and it has been demonstrated recently that replication of OriP-containing plasmids is indeed dependent on the chromosomal initiation factors, ORC2 and Cdt1. On the other hand, in the viral productive cycle, the EBV genome is amplified 100- to 1000-fold by the viral replication machinery. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments and the lytic programme arrests cell cycle progression and changes the cellular environment greatly. It has been revealed recently that the EBV lytic programme promotes an S-phase like cellular condition, which most favours viral lytic replication. This review describes recent advances regarding the molecular basis of EBV DNA replication during latent and lytic infections and then refers to cellular circumstances after induction of the lytic replication of EBV. Based on the molecular mechanism for the EBV lifestyle, purposeful induction of the lytic form of EBV infection is now advocated as one of the strategies for specific destruction of Epstein-Barr virus (EBV)-associated malignancies where the virus is latently infected.

Cell Cycle↗
PubMed1998

Kaposi sarcoma-associated herpesvirus/human herpesvirus 8, cytokines, growth factors and HIV in pathogenesis of Kaposi's sarcoma.

Epidemiological studies have strengthened the case for Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 being the long-sought Kaposi sarcoma agent, but have also pointed to a role for other co-factors. Like other tumour viruses, Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 establishes a latent (persistent) infection in Kaposi sarcoma-spindle (tumorous) cells, but can also undergo lytic replication in these and other cell types. Several latent and lytic viral genes may play a role in the pathogenesis of Kaposi's sarcoma. Although Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 contains at least two genes with transforming properties, it has not yet been shown to be oncogenic in animals. This, and other studies on inflammatory/angiogenic cellular and viral cytokines as well as HIV-Tat, emphasizes the multifactorial complexity of the pathogenesis of Kaposi's sarcoma.

Journal Article↗