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PubMed1987

Specific stimulation of simian virus 40 late transcription in vitro by a cellular factor binding the simian virus 40 21-base-pair repeat promoter element.

We have identified a cellular transcription factor from uninfected HeLa cells that stimulates the simian virus 40 (SV40) late mode of transcription and specifically binds the SV40 21-base-pair repeat promoter element. In particular, the late SV40 transcription factor (LSF) stimulates transcription at initiation sites L325 and L264 of the SV40 late promoter, which are the major transcription sites utilized after DNA replication during the SV40 lytic cycle. In addition, LSF appears to stimulate transcription to a lesser extent from the late-early initiation site of the early promoter. LSF binds specifically to the 21-base-pair repeats of the SV40 promoters, forming specific protein-DNA complexes, which migrate more rapidly through nondenaturing polyacrylamide gels than that formed by the previously identified transcription factor Sp1. Thus, LSF is distinguishable from Sp1 in both its transcriptional and DNA-binding properties. These findings suggest a potential role of LSF in the early to late transcriptional switch during a SV40 lytic infection.

Binding Sites↗
PubMed1995

Restoration of the Epstein-Barr virus ZEBRA protein's capacity to disrupt latency by the addition of heterologous activation regions.

The ZEBRA protein has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. ZEBRA is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the ZEBRA/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the ZEBRA domain). The ZEBRA activation region was replaced with the potent acid activation region from the herpes simplex virus VP16 protein or with the activation region of the EBV R protein. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than ZEBRA itself. Activation was not target- or cell-type dependent, nor was it dependent on the presence of virus. These activation domains restored ZEBRA's ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of ZEBRA are not dependent upon the nature of the activation domain present within ZEBRA.

B-Lymphocytes↗
PubMed1979

Late transcription and simultaneous replication of simian adenovirus 7 DNA as revealed by spreading lytically infected cell cultures.

Miller's technique of spreading DNA was applied to monkey cells productively infected with simian adenovirus 7. This permitted the visualization of cellular DNA transcription, both nucleolar and non-nucleolar, and of late transcription and replication of virus. Virus double-stranded DNA, thin fibres with very few nucleosome-like particles, were observed carrying either transcription or replication complexes. In addition, both RNP transcripts and replication forks were found on some virus duplex DNA. Virus single-stranded DNA replicative intermediates were identified on the basis of their increased thickness and contrast which results from the presence of a DNA binding protein.

Adenoviridae↗
PubMedDate not supplied

ClpP/ClpX-mediated degradation of the bacteriophage lambda O protein and regulation of lambda phage and lambda plasmid replication.

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1994

A new gene of bacteriophage P4 that controls DNA replication.

Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.

Amino Acid Sequence↗
PubMed2026

Spontaneous lytic reactivation drives a persistent B cell-vector pathway for epithelial dissemination of the Epstein-Barr virus.

The Epstein-Barr virus (EBV) establishes lifelong B cell infection via oral transmission; however, it paradoxically drives carcinomas in anatomically distant organs with striking geographic disparities. While genomic studies frequently link specific EBV variants to these epithelial cancers, the mechanisms bridging ubiquitous infection to distant, strain-dependent malignancies remain largely unresolved. Using an induction-free primary B cell system, we identify a circulating B cell-vector pathway driving immortalized epithelial dissemination. We demonstrate that B cells infected with carcinoma-associated strains exhibit markedly higher epithelial transmission compared with those carrying lymphoid strains. This contact-dependent process requires spontaneous lytic reactivation, viral DNA replication, and de novo virion production. Crucially, those infected B cells retain their transmission capacity for months, supporting sustained epithelial seeding. Mechanistically, entry requires gH/gL engagement of EphA2/desmocollin-2 (DSC2), with actin- and PI3K-dependent endocytosis. These findings define a lytic-coupled, receptor-dependent pathway by which the EBV exploits B cells to access the epithelium, offering a mechanistic framework for understanding strain tropism and host-virus interactions.

B cell vector↗
PubMed1993

Epstein-Barr virus and its interaction with the host.

Epstein-Barr virus (EBV) as a member of the herpesvirus family persists lifelong in the human body and causes diseases associated with virus replication (infectious mononucleosis, oral hairy leukoplakia) as well as neoplastic conditions such as nasopharyngeal carcinoma, B-cell lymphoma, Hodgkin's disease associated with viral latency. This complex biology relates to a highly regulated control of the persisting virus. Still, EBV is lytically produced in certain compartments of the human body. Epithelial cells were found to be of key importance for this. Various routes (cell fusion, IgA receptor-mediated uptake) were described for EBV to enter epithelial cells in the absence of CR2 receptor. Viral entry into cells, however, via CR2 receptor fusion or IgA mediated was not found to be sufficient for viral production. The molecular mechanisms for the lack of viral production in most target cells are primarily the presence of silencer activities and the early elimination of cells entering the lytic cycle. Only terminally differentiated epithelial cells are capable of supporting an efficient lytic cycle of EBV replication. EBV-mediated suppression of apoptosis as well as down-regulation of cellular and viral gene products, such as HLA molecules, which mediate recognition by the immune system, are important contributing factors to the development of these neoplasias where viral genes, possibly via interaction with anti-oncogenes, such as p53, in context with genetic and environmental factors play a key role. Novel diagnostic tools and a vaccine have been developed which could help to control EBV-related diseases.

Animals↗
PubMed1992

Presence of the diffuse early antigen of Epstein-Barr virus in lymphomas and lymphoproliferative disorders.

The authors recently demonstrated that 40% of Epstein-Barr virus (EBV) associated lymphoproliferative lesions contained lytic as well as latent EBV DNA. To examine more fully the replicative state of EBV in these disorders, the authors studied protein extracts of EBV-associated lymphoid lesions from 13 patients, most of whom were immunosuppressed, for expression of the diffuse early antigen (EA-D) of EBV, by immunoblotting techniques. The reagent used was a mouse monoclonal antibody. Seven of thirteen samples (54%) contained EA-D. These data indicate that in EBV-associated lymphoproliferative lesions, lytic viral replication occurs frequently, manifested by the presence of EBV diffuse early antigen as well as by the presence of lytic EBV DNA replication.

Antigens, Viral↗
PubMed1986

The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.

A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.

Bacteriophage phi X 174↗
PubMed1993

Site-specific initiation of DNA replication in metazoan chromosomes and the role of nuclear organization.

We have asked whether or not Xenopus eggs or egg extracts, which have previously been shown to replicate essentially any DNA molecule, will preferentially utilize a known mammalian OBR. Our results reveal that Xenopus egg extracts can preferentially initiate DNA replication at sites chosen in vivo by the hamster cell, provided that the DNA substrate is presented to the extract in the form of a nucleus rather than bare DNA. Thus, site-specific initiation of DNA replication in metazoan cell chromosomes appears to be determined by nuclear organization as well as DNA sequence. We have also considered whether or not BPV, which was previously reported to regulate its copy number through negative as well as positive cis-acting sequences, provides a suitable paradigm for cellular origins. The BPV genome was found to contain cis-acting sequences that can suppress DNA replication driven by a lytic virus such as PyV. However, this suppression did not require any BPV protein, did not limit PyV origin activity to one initiation event per S phase, and did not affect BPV origin activity. These results, together with data from other laboratories, strongly suggest that BPV is simply a slow-replicating version of SV40 and PyV and therefore is not an appropriate model to explain how initiation of cellular DNA replication is limited to once per cell cycle.

Animals↗
PubMed1986

Expression of the late gene of simian virus 40 under the control of the simian virus 40 early-region promoter in monkey and mouse cells.

We constructed a recombinant plasmid (pVNR4) with the simian virus 40 (SV40) early promoter positioned 30 nucleotides upstream from the major SV40 late transcription initiation site at residue 325. After transfection of the recombinant plasmid DNA into COS and mouse L cells, the transcripts of the SV40 late region were analyzed by S1 nuclease and primer extension analysis. The following are the principal findings. (i) The 16S and 19S late RNAs used the characteristic wild-type splice; no detectable levels of 19S unspliced RNA were observed. (ii) The majority of the late RNAs were heterogeneous and initiated in the early region (upstream and downstream from the Hogness-Goldberg sequence), and a minor population initiated at residue 325, the principal 5' terminus of the wild-type late RNA. (iii) During SV40 lytic infection there was a shift in initiation sites used to transcribe the early region from sites that are downstream to sites which are upstream (up RNA) of the origin of DNA replication. We observed that unlike lytic infection, T antigen and viral DNA replication were not needed for the appearance of up RNA in mouse L cells. (iv) In mouse L cells late RNAs were made, and the residue 325 5' end was utilized in the absence of T antigen or DNA replication. (v) In COS cells we found down RNA and up RNA transcribed from the extrachromosomally replicating plasmid but only down RNA produced by the integrated SV40 genome.

Animals↗
PubMed1999

Hydrocortisone activation of human herpesvirus 8 viral DNA replication and gene expression in vitro.

BACKGROUND: Patients undergoing chronic steroid therapy for organ transplantation are at increased risk for development of human herpes virus 8(HHV-8)-associated Kaposi's sarcoma (KS). It has also been reported that following steroid withdrawal, KS lesions often undergo partial or complete regression. METHODS: We have examined the effect of corticosteroid treatment on HHV-8 replication, gene expression, and lytic protein expression in BCBL-1 cells in vitro. BCBL-1 cells were collected after culture for 24-72 hr with hydrocortisone (HC) 1-5 microM, phorbol ester 20 ng/ml (positive control), and culture medium only (negative control). HHV-8 genomic conformation was examined by Gardella gel analysis. mRNA expression of viral cyclin (v-Cyc), viral Bcl-2 (v-Bcl-2), viral macrophage inflammatory protein-I (v-MIP-I), viral interferon regulatory factor-1(v-IRF-1), and viral tegument protein (TP) was examined by RT-PCR Southern blot. Viral protein expression within the cells was examined by indirect immunofluorescence using 5 different HHV-8 positive antisera from 4 renal transplant recipients and 1 patient with classic KS. RESULTS: Gardella gel analysis revealed that HC induced an accumulation of the linear replicative genomic form of the virus in a time-dependent fashion. Southern blot analysis of the RT-PCR products revealed that HC induced increased expression of v-IRF-1, v-Bcl-2, and TP mRNA, with little discernible effect on v-Cyc, and v-MIP-I. Immunofluorescence revealed that HC induced increased numbers of cells expressing lytic antigens. CONCLUSIONS: These data indicate that hydrocortisone acts directly on BCBL-1 cells to activate the lytic cycle of HHV-8 and provide further support for the hypothesis that HHV-8 is activated in corticosteroid-treated immunocompromised patients.

DNA Replication↗
PubMed2004

Early stage of Epstein-Barr virus lytic infection leading to the "starry sky" pattern formation in endemic Burkitt lymphoma.

CONTEXT: Burkitt lymphoma (BL) is histologically characterized by a "starry sky" appearance, representing scattered macrophages that have phagocytosed cell debris among proliferating lymphoma cells. As is well known, almost all the neoplastic cells of endemic BL are infected with Epstein-Barr virus (EBV). Previous studies have indicated that most of the EBV in B cells is latent, and few virus particles enter the lytic cycle. OBJECTIVE: To examine the histologic relationship between EBV infection stages and the formation of the starry sky pattern in African endemic BL tissues. DESIGN: Tissue samples from 44 patients with African endemic BL were examined with immunohistochemistry and in situ hybridization. We used EBV-encoded small RNA (EBER) as a marker of latent infection, and BamHI H left frame 1 (BHLF1) and BamHI Z EBV replication activator (ZEBRA) as lytic cycle markers. RESULTS: In all cases, signals for EBER were found in most neoplastic lymphocytes, and in 73% of cases, signals for BHLF1 and/or ZEBRA were recognized in the lymphoma cells within and around the lacunae in starry sky figures. The mean number of lacunae per unit area in cases positive for lytic cycle markers was significantly higher than that in negative cases (P <.001). CONCLUSIONS: Our findings suggest that EBV-infected lymphoma cells in the lytic cycle, which eventually lapse into cell death, are phagocytosed prior to their rupture by macrophages that have migrated into the parenchyma. We emphasize that transition of EBV-infected lymphoma cells to the lytic cycle is one of the histomorphogenetic factors influencing the formation of starry sky pattern in endemic BL.

Adolescent↗
PubMed1993

The C-terminal third of UL42, a HSV-1 DNA replication protein, is dispensable for viral growth.

UL42 is the herpes simplex virus type 1 DNA polymerase (Pol) accessory protein and is required for viral DNA replication and growth. Previous results from this laboratory demonstrated that the N-terminal two thirds of the protein contains all of the biochemical activities of the protein which can be measured in vitro. These activities include dsDNA-binding, association with DNA polymerase, and stimulation of polymerase activity. To better understand the functions of UL42 in infected cells, we have isolated and characterized two viral recombinants, UL42lacZ and n338. In the mutant virus UL42lacZ, the UL42 gene was disrupted by insertion of the Escherichia coli lacZ gene, while in the mutant virus n338, a termination codon was introduced after amino acid position 338. Analysis of the mutant phenotypes suggest that (1) the first 338 residues of UL42 retain all the functions necessary for viral DNA replication and growth in lytic infection, (2) localization of UL42 to the cell nucleus is independent of Pol, and (3) localization of ICP8 (ssDNA-binding protein) to prereplication sites is independent of functional UL42.

Animals↗
PubMed2003

Partition operon expression in the linear plasmid prophage N15 is controlled by both Sop proteins and protelomerase.

The temperate coliphage N15, unlike most low copy-number prokaryotic replicons, is maintained as a linear DNA molecule with covalently closed ends. Accurate partitioning of the plasmid prophage is assured by a close homologue of the sop locus of the F plasmid. However, the region upstream of the N15 sopAB genes contains multiple putative promoters, in contrast to F sop whose expression is driven by one negatively autoregulated promoter. In addition, the centromere of N15 is represented by four inverted repeats located at widely separated sites within the region essential for replication and control of lytic functions. We have analysed expression of N15 sop genes. We find that transcription of N15 sop is driven by two major promoters. The first, P1, is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second promoter, P2, is upstream of P1 and is several times stronger. It is insensitive to regulation by Sop proteins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These results establish a regulatory link between the partition system and other processes of N15 maintenance.

Coliphages↗
PubMed2007

Rhesus monkey rhadinovirus: a model for the study of KSHV.

Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi sarcoma-associated herpesvirus (KSHV)-a gamma-2 herpesvirus and the etiologic agent of three malignancies associated with immunosuppression. In contrast to KSHV, RRV displays robust lytic-phase growth in culture, replicating to high titer, and therefore holds promise as an effective model for studying primate gammaherpesvirus lytic gene transcription as well as virion structure, assembly, and proteomics. More recently, investigators have devised complementary latent systems of RRV infection, thereby also enabling the characterization of the more restricted latent transcriptional program. Another benefit of working with RRV as a primate gammaherpesvirus model is that its efficient lytic growth makes genetic manipulation easier than that in its human counterpart. Exploiting this quality, laboratories have already begun to generate mutant RRV, setting the stage for future work investigating the function of individual viral genes. Finally, rhesus macaques support experimental infection with RRV, providing a natural in vivo model of infection, while similar nonhuman systems have remained resistant to prolonged KSHV infection. Recently, dual infection with RRV and a strain of simian immunodeficiency virus (SIV) has led to a lymphoproliferative disorder (LPD) reminiscent of multicentric Castleman disease (MCD)--a clinical manifestation of KSHV infection in a subset of immunosuppressed patients. RRV, in short, shows a high degree of homology with KSHV yet is more amenable to experimental manipulation both in vitro and in vivo. Taken together, these qualities ensure its current position as one of the most relevant viral models of KSHV biology and infection.

Animals↗
PubMed1975

Infection of mouse blastocysts with SV40 DNA: normal development of the infected embryos and persistence of SV40-specific DNA sequences in the adult animals.

In SV40-transformed culture cells, viral-specific sequences have been found to be covalently linked to host sequences (Sambrook et al. 1968). The most appealing interpretation to explain the presence of SV40-specific sequences in adult mice following infection at the preimplantation stage would be to assume that the viral DNA was integrated at this early stage of development into the host genome and was thus conserved during further development. However, our results do not exclude an extrachromosomal existence of the SV40 genome, for example, as an independently replicating plasmid or as a lytic infection in a few permissive cells. So far our attempts to demonstrate autonomous SV40 DNA replication in early mouse embryos have been unsuccessful. We plan to investigate whether the SV40-specific information can be genetically transmitted from the infected mice to their offspring; chromosomal integration would be proven if transmission of SV40 DNA occurred in accordance with simple Mendelian expectations. The injection of mouse blastocysts with purified SV40 DNA did not detectably interfere with normal development of the embryos to healthy adult mice, which were still tumor-free at one year of age. This was not due to the trivial possibility that the viral DNA did not successfully infect and was eliminated from the injected embryos, as virus-specific DNA sequences were detected in 40% of the infected year-old animals, or in about 25% of DNA preparations extracted from some of their tissues (Table 1). It is nevertheless possible that the animals may not have been old enough to exhibit tumorigenesis of SV40 origin; to test this possibility, the experiment will have to be repeated for longer survival periods. The absence of any obvious signs of expression of viral genetic functions, i.e., tumor formation, up to one year of age of the host is reminiscent of the "cryptic transformants" described earlier (Smith et al. 1972) which harbor SV40 information but behave essentially like normal untransformed cells. Whether transcription or translation of the virus gene can occur in infected mice is presently an open question. Testing for expression of an integrated viral genome in diverse differentiated tissues may provide a useful model system to study the regulation of differentiation. These matters are currently being investigated.

Animals↗
PubMed1981

SV40-transformed simian cells support the replication of early SV40 mutants.

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.

Cell Line↗