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At least 811 records · Page 45Linked to original sources
PubMed1993

A concatenated form of Epstein-Barr viral DNA in lymphoblastoid cell lines induced by transfection with BZLF1.

The replicative form of Epstein-Barr virus (EBV) DNA was studied using two lymphoblastoid cell lines, X50-7 and 6F11, which are latently infected by Epstein-Barr virus. The lytic cycle of EBV infection was induced by transfection of the cells with the BRLF1/BZLF1 coding region of the P3HR-1 defective genome. We combined two techniques to identify the productive replicative form of Epstein-Barr viral DNA in the lytic cycle-induced cells. Restriction enzyme analysis followed by Southern blot hybridization identified a significant increase in the fused fragment encompassing both ends of EBV DNA. This indicates an increase in either episomal DNA or concatameric linear DNA. Southern blot analysis of in situ lysing gels revealed that the cellular content of linear EBV DNA was also increased significantly after the initiation of the viral lytic cycle, while the amount of circular DNA remained approximately constant. We propose from these results that the source of the fused fragment encompassing both ends of EBV DNA is a concatenated linear EBV DNA molecule, and that such a concatenated molecule most likely represents a replicative form of EBV DNA in productively infected cells.

Cell Line↗
PubMed1990

Fluorometric in vivo determination of Bacillus subtilis and phage 2C DNA.

The validity of in vivo fluorometric assays was ascertained for phage and bacterial DNA measurements. The following parameters were determined by this simple technique. The DNA content of dividing cells of Bacillus subtilis 168/2 was 2.65 times higher than in resting cells. Assuming that resting cells harbor 1 genomic equivalent, its Mr was estimated to be 4.4 x 10(9) Da. A polymerization rate during growth of 788,000 bp min-1/cell is accounted for by a multifork replication mechanism. Both phage and host DNA could be measured accurately during the lytic cycle. Phage 2C DNA synthesis proceeded at a linear rate of 5.2 genome equivalents min-1.

Bacillus subtilis↗
PubMed1999

Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression.

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

Chemokines↗
PubMed1987

Polypeptide synthesis during lytic induction of phage 11 of Staphylococcus aureus.

Staphylococcus aureus phage 11 was induced to replicate by treatment of lysogens with mitomycin C. Lysis was complete in 80 min at 37 degrees C in complete synthetic medium. Labelling with L-[3H]lysine showed a stimulation of protein synthesis during phage replication, followed after 30 min by shut-down of host protein synthesis while virion protein synthesis continued. The synthesis of approximately 16 virus-specific proteins was detected during replication, with seven of these recovered in purified virions after lysis. The remainder appeared to be synthesized earlier in phage replication and thus may represent precursors of virion proteins, or regulatory proteins, or enzymes associated with phage replication. Protein synthesis during replication of three suppressible morphological (head, tail) mutants of phage 11 did not differ significantly from that seen in the wild-type lysogen, indicating that the mutation in each case affected a protein whose synthesis was not detectable, or which was synthesized but did not fulfil its role in virion maturation or assembly. However, in a suppressible 'early' mutant which did not lyse when treated with mitomycin C, the synthesis of the 'late' (virion) proteins (with one exception) did not occur. 'Early' proteins were apparently made normally in this mutant, and there was no shut-down of host protein synthesis. This mutant phage presumably encodes a defective 'early' protein involved in the regulation of replication, or a key precursor polypeptide of 'late' protein synthesis. The morphological mutants provided a means to analyse and tentatively to allocate six of the virion proteins to head or tail/baseplate structures.

Mitomycin↗
PubMed1983

p lambda CM system: observations on the roles of transposable elements in formation and breakdown of plasmids derived from bacteriophage lambda replicons.

Transduction with phage derived from a 2-year-old lysate of lambda cam105 (lambda::Tn9) gave rise to chloramphenicol-resistant (Cm(r)) transductants harboring a plasmid (plambdaCM1) formed from lambda cam105 by a Tn9-mediated adjacent deletion to position 36.07 kilobases in the N cistron of lambda. The plambdaCM element can replicate as a plasmid, insert into the bacterial genome, or reproduce lytically as a phage on cells that provide N function. The feasibility of obtaining high titers in encapsidated form and the ease of synchronous introduction into and recovery from bacterial populations make plambdaCM very suitable for quantitative studies of recombination involving transposable elements. Replicon fusions between plambdaCM1 and RSF1596 (pMB8::Tn3Delta596) occur by duplication of either IS1 (at low rate in the absence of TnpA activity) or Tn3Delta596 (in the presence of TnpA activity). At 24 or 32 degrees C, the rate of increase of TnpA-mediated fusions per plambdaCM is about 2% per cell doubling. RSF103 contains the deleted Tn1DeltaAp (which lacks intact beta-lactamase and TnpR resolvase coding sequences) adjacent to a streptomycin resistance (Sm(r)) determinant. We observed that Tn1DeltaAp mediates insertions of external RSF103 sequences into the R388 plasmid. R388::Tn1DeltaAp plasmids show transposition immunity in cells lacking TnpR activity. Using the plambdaCM system, we isolated adjacent transpositions of the RSF103 Sm(r) determinant. The resulting plambdaCM-Sm cosmids contain Sm(r) genetic material flanked by direct repeats of Tn1DeltaAp, and all are deleted for some RSF103 or plambdaCM sequences. The plambdaCM-Sm constructs will fuse into R388 by duplication of a single Tn1DeltaAp element. In the presence of tnpR(+) (but not tnpR) Tn1 or Tn3 elements, all Tn1DeltaAp-mediated complex replicons break down completely and rapidly to simple Tn1DeltaAp inserts. The equilibrium for resolution is at least 10(5):1, and resolution is more than 90% complete after 40 min of exposure to a tnpR(+) cytoplasm. In the absence of TnpR, Rec, and Red activities, Tn1DeltaAp-mediated complex replicons yield simple Tn1DeltaAp inserts at a lower rate. The presence of intact RSF103 replication determinants between direct Tn1DeltaAp repeats appears to accelerate this precise TnpR- and Rec-independent breakdown.

Bacteriophage lambda↗
PubMed2003

Inhibition of gammaherpesvirus replication by RNA interference.

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.

Animals↗
PubMed2001

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.

Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.

Adenosine Diphosphate↗
PubMed1993

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.

Cells, Cultured↗
PubMed1993

Rates of spontaneous mutation among RNA viruses.

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses.

Bacteriophages↗
PubMed1970

Control of gene function in baceriophage T4. II. Synthes of messenger ribonucleiccid and proei after interrupting deoxyribonucleic acid replication and glucosylation.

Replication of T4 deoxyribonucleic acid (DNA) is known to be required for the onset of transcription of late T4 genes. Once late gene transcription has been initiated, DNA replication is no longer required for maintaining synthesis of late or early T4 messenger ribonucleic acid (mRNA). Late phage proteins (lysozyme and tail fibers) continue to be produced at constant rates after interrupting T4 DNA synthesis. The ability of the host cell to glucosylate the T4 progeny DNA has no demonstrable influence on the rates at which T4 mRNA and late proteins are synthesized after the interruption of DNA synthesis. To explain the requirement of T4 DNA replication for the onset of late gene transcription, we suggest that T4 DNA in a nascent state is mandatory for the initial late gene transcription, or perhaps for late gene transcription throughout the lytic cycle. T4 DNA in a nascent state could be segregated from the bulk of the replicating DNA, used only as template for RNA synthesis, and prevented from being modified by methylation, glucosylation, or maturation processes. The fact that no, or very little, nonglucosylated T4 DNA is extractable from T4LB3-infected CR63 after arresting DNA synthesis does not rule out this possibility.

Centrifugation, Density Gradient↗
PubMed2002

Expression of viral and human dUTPase in Epstein-Barr virus-associated diseases.

Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and pyrophosphate thus preventing the incorporation of uracil into replicating DNA. Previous studies of several virus models have suggested that viral dUTPases may be required for virus replication in resting cells whereas in proliferating cells cellular dUTPase may substitute for a mutant viral protein. Using monoclonal antibodies and immunohistochemistry, Epstein-Barr virus-associated non-neoplastic and neoplastic diseases were studied for the expression of viral and human dUTPases. Oral hairy leukoplakia, an AIDS-associated lesion of the tongue, is known to support EBV replication in the upper epithelial cell layers. In agreement with this, strong focal expression of EBV dUTPase was detected in the upper epithelial cell layers of oral hairy leukoplakia whereas expression of human dUTPase was confined to the basal proliferative cell compartment. Furthermore, in infectious mononucleosis tonsils, rare scattered small lymphoid cells expressed EBV dUTPase, consistent with the expression pattern of other EBV lytic cycle antigens. These findings are in agreement with the notion that EBV replicates in resting cells. Three EBV-associated tumours, Hodgkin lymphoma, Burkitt lymphoma and nasopharyngeal carcinoma, lacked detectable expression of EBV dUTPase, in agreement with the notion that EBV infection is largely latent in these tumours. By contrast, expression of human dUTPase was observed regularly in these tumours. These results suggest that EBV dUTPase may be a suitable target for anti-viral therapy and that inhibitors of human dUTPase should prove useful for the treatment of human tumours, including EBV-associated cancers.

Antibodies, Monoclonal↗
PubMed2004

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma.

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.

Animals↗
PubMed2003

Gene therapy for killing p53-negative cancer cells: use of replicating versus nonreplicating agents.

Research has focused on the use of viral vectors to attack p53-negative cancer cells. Such agents may be nonreplicating, whereas others are replicating. This paper uses mathematical models to study the conditions under which therapy can lead to tumor remission. It is found that the optimal characteristics of the vector can be quite different depending on whether the virus replicates or not. If it does not replicate, the rate of virus-induced tumor cell killing should be maximized. If the virus does replicate, the rate of virus-induced cell killing should be kept small. If the virus is too lytic in cancer cells, viral spread is compromised, resulting in persistence of both virus and tumor. This has important implications for choosing the correct techniques to evaluate replicating viruses in culture. A low multiplicity of infection must be used for evaluation, because this mimicks the spread of the virus through an established tumor. If a high multiplicity of infection is used, the virus that appears most efficient in this evaluation can be least efficient at eradicating the cancer in vivo. Theoretical results are discussed in the context of experimental data.

Animals↗
PubMed1999

Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle disease virus.

Direct infection of tumor cells with viruses transferring protective or therapeutic genes, a frequently used procedure for production of tumor vaccines in human gene therapy, is an approach which is often limited by the number of tumor cells that can reliably be infected as well as by issues of selectivity and safety. We report an efficient, selective and safe way of infecting human tumor cells with a natural virus with interesting pleiotropic immune stimulatory properties, the avian paramyxovirus Newcastle disease virus (NDV). Two of the six viral genes (HN and F) modify the tumor cell surface by introduction of new adhesion molecules for lymphocyte interactions and other viral genes stimulate host cell genes and local production of cytokines and chemokines which can recruit a broad antitumor response in vivo. A large variety of human tumor cells is shown to be efficiently infected by NDV with viral replication being independent of tumor cell proliferation. Such properties make NDV a suitable agent for modification of noncultured freshly isolated and gamma-irradiated patient-derived tumor cells. For the apathogenic non-lytic strain NDV-Ulster which is used in our clinical vaccine trials, we demonstrate selective replication in tumor cells as compared with corresponding normal cells. Furthermore, we present evidence that new virions produced by infected tumor cells are non-infectious using three different quantitative test methods. Our results demonstrate feasibility and broad applicability of this strategy of human tumor vaccine modification. Post-operative vaccination with the autologous virus-modified vaccine ATV-NDV thus provides a reasonable potential for pleiotropic modifications of the immune response of cancer patients against their own tumor.

Cancer Vaccines↗
PubMed1980

Recombinant P4 bacteriophages propagate as viable lytic phages or as autonomous plasmids in Klebsiella pneumoniae.

We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD+. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (less than or equal to 10(-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sid1) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.

Bacteriophages↗
PubMed1977

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.

Antigens, Viral↗
PubMed2005

Alternative methods to limit extracellular bacterial activity for enumeration of intracellular bacteria.

The gentamicin survival assay, a method routinely used to estimate bacterial infection of eukaryotic host cells, depends on the presumed limited penetration of gentamicin across the eukaryotic cell membrane. However, some studies have suggested that gentamicin may in fact enter eukaryotic cells and kill intracellular bacteria. In this study we devised alternative methods to enumerate intracellular Salmonellae using a lytic bacteriophage, SP6, and an amino acid auxotroph, Pro- mutant, which replicates selectively within host cells in the presence of its uptake inhibitor, 3,4-dehydro-L-proline. The conventional gentamicin survival assay was systematically compared with the alternative methods for the enumeration of intracellular Salmonellae. We found that gentamicin decreases the survival of intracellular Salmonellae when added to extracellular media at concentrations above 20 microg/ml. The alternative methods do not suffer from this disadvantage, suggesting that they should be used to replace the gentamicin survival assay. In addition, the proline auxotroph method could be applied to detect bacterial release from host cells.

Anti-Bacterial Agents↗
PubMed1976

4S oop RNA is a leader sequence for the immunity-establishment transcription in coliphage lambda.

oop RNA, which is initiated at the po promoter and is 81 nucleotides long, can function as a leader sequence for the lambda immunity establishment transcription, previously believed to originate at a special promoter pre located in the y region. Thus, oop RNA seems to have a dual role, either favouring the lytic cycle as a primer for the initiation of lambda DNA replication, or leading to the establishment of lysogeny when elongated into the imm transcript, which directs synthesis of the repressor.

Coliphages↗