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PubMed2000

Reconstitution of NK cell activity in HIV-1 infected individuals receiving antiretroviral therapy.

We studied natural immunity mediated by natural killer (NK) cells in 62 HIV-1 infected individuals, 54 HIV-1 infected individuals receiving highly active antiretroviral therapy (HAART) for more than one year and 8 HIV-1 infected individuals without antiretroviral therapy. 22 individuals had a complete suppression of viral replication characterized by viral load values <50 copies/ml, whereas 32 individuals presented with persistent viral replication. The 8 untreated patients had an indication to start antiretroviral treatment. Lytic activity of NK cells was measured in a 51chromium release assay. In patients with persistent viral replication under HAART NK cell activity was significantly decreased compared to patients with effective control of HIV viremia. Patients with complete suppression of HIV replication displayed a similar NK activity to healthy control persons. Differences in antibody-dependent cellular cytotoxicity (ADCC) were not observed. Further studies will investigate whether decreased NK cell activity is a reason for or the consequence of persistent viral replication.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1982

The rex region of bacteriophage lambda: two genes under three-way control.

The nucleotide sequence of the phage lambda rex region consists of 1428 bp and codes for two genes, rexA and rexB. Hence the complete lambda immunity region codes for four genes and covers 2664 bp of sequence unique to lambda, as defined by the left and right boundaries of the imm434 region. Coordinate expression of both rexA and rexB, which are co-regulated with the cI repressor gene from promoters p rm and p re is responsible for the Rex phenotype, i.e. exclusion of a wide variety of superinfecting phage such as T4rII. The position of a third promoter, p lit, which overlaps the carboxy-terminal end of the rexA coding region, permits expression of rexB without rexA, from the resulting 470 nucleotide lit RNA. The lit transcript, therefore, must act as messenger for rexB in the noncoordinate expression of the rex genes that occurs late in lambda lytic infection. The coordinate and noncoordinate expression of rexB and rexA suggests a dual role for the very hydrophobic rexB protein. Studies of lambda early and late DNA replication implicate rexB as having auxiliary functions in both lysogenisation and lytic infection.

Bacteriophage lambda↗
PubMed1998

Escherichia coli dnaA gene function and bacteriophage lambda replication.

Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from bacteriophage lambda has been demonstrated previously. Here, using a series of dnaA temperature-sensitive mutants, we investigated dnaA allele specificity of the replication of phages lambda P+ and lambda Pts 1 pi A66. We found that phage lambda P+ produces its progeny efficiently at 43 degrees C irrespective of the dnaA allele, whereas lambda Pts 1 pi A66, which is unable to develop lytically in the dnaA+ host at this temperature, can replicate with different efficiency in certain dnaA mutants. Since the main role of DnaA in lambda development seems to be stimulation of transcription from the pR promoter, we measured the activity of this promoter (using a pR-lacZ fusion) and the abundance of pR-derived transcripts (by Northern blotting analysis) in dnaA+ host and dnaA(ts) mutants at 30 and 43 degrees C. We found significant differences in the activity of pR in various dnaA(ts) mutants at 30 degrees C, which indicate different levels of stimulation of this promoter by products of particular dnaA alleles at permissive temperature. Differential levels of DnaA-mediated stimulation of pR in various dnaA(ts) mutants were also found at 43 degrees C. Stimulation of the pR promoter by DnaA is necessary for both efficient production of the lambda replication proteins, O and P, and effective transcriptional activation of ori lambda. The differences in the efficiency of pR activation observed in dnaA mutants at 30 and 43 degrees C can explain the mechanisms of allele specificity of dnaA gene function in the replication of bacteriophage lambda and plasmids derived from this phage.

Alleles↗
PubMed1986

Transcriptional control of herpesvirus gene expression: gene functions required for positive and negative regulation.

We have used an in vitro nuclear run-off assay to measure the levels of transcription of specific herpes simplex virus genes at different times during a lytic infection. We analyzed the effects of inhibition of DNA replication and of defects in two herpes simplex virus regulatory proteins on the transcription of these genes. We present evidence that the transcription of the alpha ICP4 gene is negatively regulated during a lytic infection. The regulation of ICP4 gene transcription requires the beta protein ICP8 (where ICP = infected cell polypeptide). Transcription of the beta ICP8, gamma 1 ICP5, and gamma 2 glycoprotein C (gC) genes was dependent on ICP4, and transcription of the gamma 2gC gene was strongly inhibited when DNA replication was blocked. Defects in ICP8 also resulted in increased levels of transcription of the ICP4, ICP8, ICP5, and gC genes from parental viral genomes. Our results suggest that ICP8 may be important in maintaining the highly ordered cascade of viral gene expression.

DNA Replication↗
PubMed2000

Expression of Epstein-Barr virus lytic gene BRLF1 in nasopharyngeal carcinoma: potential use in diagnosis.

Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein-Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT-PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83.0%), but only in 1 of 53 control samples (1.9%). Furthermore, the antibody binding regions were found in the C-terminal two-thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC.

Antibody Specificity↗
PubMedDate not supplied

[A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions].

The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation.

Animals↗
PubMed1994

Epstein-Barr virus latent and replicative gene expression in post-transplant lymphoproliferative disorders and AIDS-related non-Hodgkin's lymphomas. French Study Group of Pathology for HIV-associated Tumors.

In acquired immunodeficiency, B-cell proliferation is usually associated with Epstein-Barr virus (EBV), implying the impairment of the normal control of EBV and EBV-infected cells. It has been assumed that EBV infection is latent in lymphoproliferative disorders. In order to determine the type of latency and to investigate whether any lymphoproliferative disorders enter into the lytic cycle, we analyzed the expression of latent and replicative EBV genes in 9 post-transplant lymphoproliferative disorders (PTLD) and in 23 EBV-positive AIDS-related non-Hodgkin's lymphomas (AR-NHL). The PTLD cases were categorized into polyclonal or monoclonal polymorphic tumors and monoclonal monomorphic tumors. The AR-NHL cases included large-cell/immunoblastic (LC/IB) and Burkitt's lymphoma (BL) groups. We demonstrated that varying patterns of latent-viral-gene expression are exhibited showing the 3 forms of latency. Polymorphic PTLD and LC/IB AR-NHL frequently expressed type II or III latency, whereas monomorphic tumors and BL AR-NHL showed type I latency. It is noteworthy that 3 cases of BL AR-NHL expressed latency II form. Induction of lytic cycle highlighted by the expression of BZLF1 occurred in 55.5% of PTLD, 36% of LC/IB and 22% of BL AR-NHL. In contrast, late viral proteins indicating productive cycle were present in 22% of PTLD, 14% of LC/IB, and were absent in BL cases. These data suggest that the impairment of EBV control permits disruption of latency, but the initiation of the lytic cycle may not always lead to viral production.

Gene Expression↗
PubMed1984

Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication.

Chimeric SV40 DNA containing only the early region, or plasmid DNA harboring the origin-promoter-enhancer region of SV40, when introduced into CV-1 or Cos-1 monkey cells by DEAE-dextran mediated transfer are rapidly assembled in a typical chromatin structure revealed by the generation of a regular 190 bp repeat ladder after micrococcal nuclease digestion. DNA replication is not required for this assembly process. Chromatin-specific DNase I hypersensitive sites are observed in the enhancer region of these minichromosomes. The pattern of the sites differs between non-replicating and post-replicated chromatin. The latter is identical to that observed in the lytic cycle. The presence of large T antigen is not sufficient for the shift in the structure of the chromatin. These experiments suggest that replication can modulate protein-DNA interactions during viral infection or upon cell differentiation.

Animals↗
PubMed1993

The lytic origin of herpesvirus papio is highly homologous to Epstein-Barr virus ori-Lyt: evolutionary conservation of transcriptional activation and replication signals.

Herpesvirus papio (HVP) is a B-lymphotropic baboon virus with an estimated 40% homology to Epstein-Barr virus (EBV). We have cloned and sequenced ori-Lyt of herpesvirus papio and found a striking degree of nucleotide homology (89%) with ori-Lyt of EBV. Transcriptional elements form an integral part of EBV ori-Lyt. The promoter and enhancer domains of EBV ori-Lyt are conserved in herpesvirus papio. The EBV ori-Lyt promoter contains four binding sites for the EBV lytic cycle transactivator Zta, and the enhancer includes one Zta and two Rta response elements. All five of the Zta response elements and one of the Rta motifs are conserved in HVP ori-Lyt, and the HVP DS-L leftward promoter and the enhancer were activated in transient transfection assays by the EBV Zta and Rta transactivators. The EBV ori-Lyt enhancer contains a palindromic sequence, GGTCAGCTGACC, centered on a PvuII restriction site. This sequence, with a single base change, is also present in the HVP ori-Lyt enhancer. DNase I footprinting demonstrated that the PvuII sequence was bound by a protein present in a Raji nuclear extract. Mobility shift and competition assays using oligonucleotide probes identified this sequence as a binding site for the cellular transcription factor MLTF. Mutagenesis of the binding site indicated that MLTF contributes significantly to the constitutive activity of the ori-Lyt enhancer. The high degree of conservation of cis-acting signal sequences in HVP ori-Lyt was further emphasized by the finding that an HVP ori-Lyt-containing plasmid was replicated in Vero cells by a set of cotransfected EBV replication genes. The central domain of EBV ori-Lyt contains two related AT-rich palindromes, one of which is partially duplicated in the HVP sequence. The AT-rich palindromes are functionally important cis-acting motifs. Deletion of these palindromes severely diminished replication of an ori-Lyt target plasmid.

Animals↗
PubMed1990

Biological and molecular aspects on herpes simplex virus latency.

Latent reactivateable herpes simplex virus (HSV) infections of sensory neurons of peripheral ganglia are most plausibly the source of clinical herpetic recurrences. The establishment of latency is the result of a concert of both viral and cellular factors such as the neuroinvasiveness of the virus, the relative density of receptors binding virus to the nerve cell plasma membrane, the permissiveness of the infection, including the axonal transport of the viral nucleocapsids, and the restriction of virus replication. Several hypotheses have been presented suggesting various mechanisms for the restriction of the HSV infection of the neuron. Thus, for instance the the importance of thymidine kinase negative mutants, hypermethylation of viral DNA and the existence of latency-associated viral genes have been discussed. Activation of the latent infection to a virus-producing lytic infection by means of superinfection with a replication-incompetent mutant is probably a result of genetic complementation. Reactivation by means of superinfection with replication-competent virus seems dependent upon the multiplicity of the superinfecting virus and more than one copy of the virus genome is required for the initiation of the reactivating process. These observations would be consistent with the overcoming of a cellularly controlled restriction of the latent infection. The cellular control of latency which can be impaired mechanically and chemically seems particularly important for the maintenance of latent HSV infection. However, recent observations indicate that reactivation of latent HSV infection is also associated with the expression of a latency-related viral gene (LAT), whereas establishment of the latency apparently is influenced by other properties determined by the genome of the virus, as well as by the capacity of the cell to restrict the lytic infection.

Ganglia↗
PubMed2006

Trimethylation of histone H3 lysine 4 by Set1 in the lytic infection of human herpes simplex virus 1.

Human herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that causes facial, ocular, and encephalitic disease in humans. Previous work showed that the genome of HSV-1 is associated with acetylated and methylated histones during lytic infection. However, the physiological role of histone modifications in lytic infection of HSV-1 is unclear. We examined the role of protein methylation in lytic infection of HSV-1 using a protein methylation inhibitor, 5'-deoxy-5'-methylthioadenosine (MTA). We found that MTA strongly reduces the transcription and replication of HSV-1. Moreover, MTA treatment decreases the level of trimethylation of lysine 4 in histone H3 (H3K4me3) on the HSV-1 genome. These results suggest that protein methylation, and in particular, histone methylation, is involved in the lytic infection of HSV-1. To delineate the underlying mechanism, we investigated the role of two H3K4 methyltransferases, Set1 and Set7/9, in the lytic infection of HSV-1. Using small interference RNA, we found that the reduction of Set1, but not Set7/9, reduces the transcription and replication of HSV-1 and specifically decreases H3K4me3 on the virus genome. These results indicate that H3K4me3 mediated by Set1 is required for optimal gene expression and replication of HSV-1 during lytic infection and suggest that this pathway could be a potential point of pharmacological intervention during HSV-1 infection.

Gene Expression Regulation, Viral↗
PubMed1987

Modulation of cell wall synthesis by DNA replication in Escherichia coli during initiation of cell growth.

Resting cells of Escherichia coli are able to initiate growth and murein biosynthesis in the presence of beta-lactam antibiotics binding to penicillin-binding proteins (PBPs) 1a and 1b (E. J. de la Rosa, M. A. de Pedro, and D. Vázquez, Proc. Natl. Acad. Sci. USA 82:5632-5635, 1985). Under these conditions, cells elongate normally until they approach the first doubling in mass, the time at which cell lysis starts. Assuming that coupling between DNA replication and cell division both in cells starting growth and in growing cells is essentially similar, triggering of the lytic response in the beta-lactam-treated cells coincides with the termination of the first round of DNA replication. This coincidence suggests that both events are interrelated. We investigated this possibility by studying the initiation of growth in cultures of wild-type strains and in cell division mutants treated with beta-lactams inhibiting PBPs 1a and 1b and with the DNA replication inhibitor nalidixic acid. Addition of nalidixic acid, even late in the first cell cycle, prevented the lytic response of the cells to the blockade of PBPs 1a and 1b. The effect of nalidixic acid is more likely due to its action on DNA replication itself than to its indirect inhibitory effect on cell division or to its ability to induce the SOS system of the cell. These observations favor the idea that the cell wall biosynthetic machinery might be modulated by DNA replication at precise periods during cell growth.

Bacterial Proteins↗
PubMed2002

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Antineoplastic Agents↗
PubMed2004

Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2.

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P(E)1 abolished origin function. Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P(E)1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

Bacteriophages↗
PubMed1995

Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays.

Using a transient replication assay in which cosmid DNAs were cotransfected into Vero cells, we had previously demonstrated that oriLyt replication required six Epstein-Barr virus (EBV)-encoded replication genes. No oriLyt origin-binding protein was identified in this study, but oriLyt replication in the cotransfection assay was also dependent on the three lytic cycle transactivators Zta, Rta, and Mta and an activity encoded by the EBV Sal/I F fragment. We have now used expression plasmids for the six known replication proteins to further examine the question of the requirement for an oriLyt origin-binding protein. The activity in Sal/I-F was shown to be encoded by BKRF3. The predicted product of this open reading frame is an enzyme, uracyl DNA glycosylase, not an origin-binding protein, and is dispensable for replication in assays using expression plasmids. BBLF2, which is positionally related to the gene for the herpes simplex virus (HSV) UL9 origin-binding protein, was confirmed to be expressed as a spliced transcript with BBLF3 and not as an independent product. Examination of the requirement for the EBV transactivators revealed that Rta, while contributing to replication efficiency, was dispensable. Mta could be substituted by HSV IE63, and in complementation experiments with HSV replication genes, Mta was no longer required for replication of EBV oriLyt, suggesting that the contribution of Mta to replication may be indirect. Zta continued to be required for detectable oriLyt replication both with the EBV replication proteins and in the complementation assays with HSV replication proteins. We conclude that EBV does not encode an equivalent of HSV UL9 and that Zta is the sole virally encoded protein serving an essential origin-binding function.

Animals↗
PubMed1993

Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection.

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.

Apoptosis↗
PubMed1991

Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.

Animals↗
PubMed1971

Structure of replicating simian virus 40 deoxyribonucleic acid molecules.

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.

Animals↗