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PubMed1973

Effect of lysozyme on enterococcal viability in low ionic environments.

The action of lysozyme on the enterococcal cell differed markedly as a function of the ionic strength of the environment. In high ionic environments (I approximately 0.3), the traditionally slow lytic response and decrease in viability were noted. In a low ionic environment the majority of the cell wall was hydrolyzed, but cellular integrity was preserved and almost all cellular protein, deoxyribonucleic acid and ribonucleic acid remained with the lysozyme-cell complex. However, under these conditions, lysozyme inactivated energy-yielding metabolism, and a rapid extensive loss of viability was observed. Some other basic compounds without lytic activity on the cell wall also effected a substantial reduction in viability. The data suggest that lysozyme acts on the cell membrane to effect disruption of cellular metabolism.

Bacterial Proteins↗
PubMed1984

Phasmid P4: manipulation of plasmid copy number and induction from the integrated state.

"Phasmid" P4 is unusual in that it is capable of (i) temperate, (ii) lytic, helper-dependent, and (iii) plasmid modes of propagation. In this report we characterize most of the known P4 genetic functions as to their essential or nonessential roles in the stable maintenance of plasmid P4 vir1 (pP4 vir1 (pP4 vir1). We also identify growth conditions that can be used to stably maintain pP4 vir1 at any one of several different copy number levels (n = 1 to 3, n = 10 to 15, or n = 30 to 40). Analyses of a temperature-sensitive alpha derivative of pP4 vir1 show that shifting the temperature from 37 to 42 degrees C allows this mutant to maintain an integrated copy of the plasmid, whereas replication of free copies is repressed because of the nonpermissive condition for their DNA synthesis. Conversely, a shift from 42 to 37 degrees C can be used to reinstate plasmid propagation. The utility of the inducible states of pP4 vir1 is discussed with respect to its attributes as a vector with the potential for cloning inserts of DNA up to 33,000 base pairs in a wide range of bacterial hosts.

Coliphages↗
PubMed2004

Discovery of mammalian genes that participate in virus infection.

BACKGROUND: Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. RESULTS: Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 x 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle. CONCLUSIONS: Tagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies.

Adenocarcinoma↗
PubMed2006

Effect of extremely low frequency electromagnetic fields (ELF-EMF) on Kaposi's sarcoma-associated herpes virus in BCBL-1 cells.

Association between extremely low frequency electromagnetic fields (ELF-EMF) and human cancers is controversial, and few studies have been conducted on their influence on oncogenic viruses. We studied the effects of 1 mT, 50 Hz sine waves, applied for 24-72 h, on Kaposi's sarcoma (KS)-associated herpesvirus (KSHV or HHV-8) in BCBL-1, a latently infected primary effusion lymphoma (PEL) cell line. ELF-EMF exposure did not affect the growth and viability of BCBL-1 cells, either stimulated or not with TPA. The total amount of KSHV DNA detected in ELF-EMF exposed cultures not stimulated with TPA did not differ from that of the unexposed controls (P = ns). However, in the presence of TPA stimulation, total KSHV DNA content was found higher in ELF-EMF exposed than in control BCBL-1 cultures (P = .024) at 72 h exposure, but not earlier. Viral DNA increase significantly correlated with increased mean fluorescence intensity/cell for the lytic antigen gp K8.1A/B (P < .01), but not with percentage of gp K8.1A/B-positive cells or of cells containing virions. Viral progeny produced under ELF-EMF exposure consisted mainly of defective viral particles.

Cell Line, Tumor↗
PubMed1997

Advances in adenoviral vectors: from genetic engineering to their biology.

Ad2 and Ad5 belong to a group of human cytolytic viruses that target the respiratory airways for reproduction, whereas latent infections establish within other tissues. Signals therefore exist that control this dichotomic process in different cell types, perhaps including cis and/or trans elements of viral origin. Since 1993, Ad2- and Ad5-based adenoviruses lacking all or part of the E1 regulatory region have been undergoing evaluation in phase I trials that target cancer and cystic fibrosis. These viruses are extremely attenuated and actually do not reproduce in most human cells. However, they retain most of the virus genetic program and often promote a significant cytotoxicity after infection, emphasizing the need to further cripple the virus biology to extend the duration of transgene expression, if required. We will review the strategies currently followed to engineer a professional lytic virus for epithelial cells into an innocuous gene delivery vehicle. Potential effects on the transducing properties of the vector that may result from the inactivation of viral activities that normally allow/regulate extrachromosomal gene expression during wild-type infection are discussed.

Adenovirus E1 Proteins↗
PubMed1996

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.

Animals↗
PubMed2005

An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate.

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.

Base Sequence↗
PubMed2006

Perforin, Granzyme B, and fas ligand for molecular diagnosis of acute renal-allograft rejection: analyses on serial biopsies suggest methodological issues.

BACKGROUND: The Perforin-Granzyme B and Fas/Fas Ligand apoptotic mechanisms are involved in the development of acute renal rejection (AR). We describe our experience of analyzing the expression of cytotoxic T-lymphotoxins (CTL) in biopsies and peripheral blood leukocytes (PBL) for the diagnosis of AR. METHODS: We analyzed Perforin (P), Granzyme B (GB) and Fas Ligand (FL) expression in 68 renal biopsies and 64 PBL using comparative kinetic RT-PCR and, for GAPDH and FL, we also replicated with real-time RT-PCR. The levels of expression were measured in different groups, such as T0 (biopsies before reperfusion and PBL in recipient before the transplant [Tx]), Td (biopsies and PBL collected for clinical purposes) and Tp (biopsies and PBL two months after Tx). RESULTS: A higher CTL expression was seen in non-rejecting (NR) biopsies in the first 2 months after Tx. P and FL were significantly more expressed during AR in all biopsies and in Td, while P remained upregulated in Tp. In PBL, there was no significant increase in CTL transcription during AR. A variable expression of CTL emerged in all T0 biopsies. CONCLUSIONS: Two lytic pathways are activated in biopsies when AR occurs shortly after Tx, whereas the P/GB mechanism prevails if it occurs later on. Only P and FL in biopsies might be able to predict AR diagnosis, but with a considerable variability in each sample, possibly due to the small portion of tissue core, which may be inadequate for molecular diagnosis. CTL expression in PBL does not correlate with histological AR.

Acute Disease↗
PubMed1975

Specific and nonspecific antitumor immunity. I. Description of an in vitro assay based on inhibition of DNA synthesis in tumor cells.

As in vitro assay of cell-mediated antitumor immunity based on the inhibition of tumor cell DNA synthesis (IDS) was devised. It was reasoned that both cytolytic and cytostatic antitumor effects could be measured in a quantitative yet generalized manner with this approach. By the use of microculture techniques and simplified methods for the determination of tritiated-thymidine incorporation by all cells in individual cultures, normal and immune effector (E) cell populations were compared in their ability to inhibit the DNA synthesis of a fixed initial number of various target tumor cells. Doubling dilutions of E cells were used to titrate the antitumor effects of normal and immune cells at many E/T (effector to target) ratios. Under conditions of alloimmunization, significant immunologically specific IDS effects could routinely be detected at an E/T ratio of less than 1 or 0.1:1, and under certain conditions at 1:100 or less. Results were highly reproducible with respect to the individual E cell donors, replicate cultures, and repeat experiments. The effects were proportional to visually determined cell destruction and independent of obvious culture artifacts. The IDS method was compared with the 51Cr release technique under various experimental conditions. The results demonstrated that decreases in E/T ratio and/or the addition of excess nonimmune cells to immune effector populations had a similar effect in both assays, which was to decrease the magnitude of the immune cell activity. Addition of excess normal cells reduced the activity of immune cells to a level below that of an equal number of immune cells tested at the same E/T ratio without added nonimmune cells. Both assays detected primarily a T lymphocyte-mediated lytic event when effectors generated by the described alloimmunizations were used. The IDS assay also detected a weak non-T-cell activity in anti-theta plus complement-treated alloimmune spleen. The possibility that this represented antibody-dependent cell-mediated cytotoxicity was raised by the finding that normal spleen ceels plus antitarget antibody had significant activity in the IDS system. The sensitivities of the two methods were compared and the potential of the IDS method was evaluated.

Animals↗
PubMed1975

Chromatin-like structures in polyoma virus and simian virus 10 lytic cycle.

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.

Cell Nucleus↗
PubMed1991

Enteric adenovirus type 40: complementation of the E4 defect in Ad2 dl808.

The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 dl808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements dl808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 dl808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

Adenovirus Early Proteins↗
PubMed1983

Polyomavirus large T antigen binds independently to multiple, unique regions on the viral genome.

To map the polyomavirus large T antigen binding sites on the viral genome we employed a quantitative immunoassay. Defined, radiolabeled fragments of the viral genome were reacted with crude nuclear extracts prepared from lytically infected mouse 3T6 cells, and the fragments bound by large T antigen were immunoprecipitated with anti-T serum and Formalin-fixed Staphylococcus aureus. The immunoprecipitated DNA was then analyzed by gel electrophoresis and autoradiography. By the use of a variety of restriction endonuclease-generated fragments of wild-type and mutant viral DNAs, the region of high-affinity binding was localized to a 153-base-pair stretch between nucleotides 5292 and 152. At least two independent binding sites lie within this region, one upstream and the other downstream of the Bg/I site at nucleotide 87. One of the binding sites is located within sequences required in cis for DNA replication; the other overlaps the TATA box and cap sites of the early transcription unit. The two sites share a common sequence, A/TGAGGC-N4/5-A/TGAGGC, which may serve as the recognition sequence for large T antigen.

Antigens, Viral, Tumor↗
PubMed2006

Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression.

This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past EBV infection and no EBV infection) and from immunosuppressed patients (30 asymptomatic renal transplant recipients and 4 liver transplant patients with diagnosed post-transplant lymphoproliferative disorders [PTLD]). Our two multiplex-nested RT-PCR assays provide a reliable, rapid and sensitive system, enabling the simultaneous detection and identification of seven latent/immortalizing and lytic EBV transcripts. These assays could be employed in further investigations in order to evaluate the EBV transcriptional profile in EBV-related diseases both in immunocompetent and immunocompromised hosts.

Adult↗
PubMed1993

Phi X174 E complements lambda S and R dysfunction for host cell lysis.

Hybrid lambda phages which have the E lysis gene of the bacteriophage phi X174 in cis to defective nonsense and deletion alleles of the normal lambda lysis genes S and R have been constructed and shown to be fully competent for plaque-forming ability, which demonstrates that the single-gene, lysozyme-independent lysis system of phi X174 and related phages can serve the lytic function for large complex phages. These hybrid phages are unable to form plaques on a slyD host. Moreover, plaque morphology indicates that in E-mediated lysis the soluble lambda R endolysin can participate in lysis, indicating that the protein E-mediated lesions are not completely sealed off from the periplasm.

Bacteriophage lambda↗
PubMed1991

Localization of the adenovirus early region 1B 55-kilodalton protein during lytic infection: association with nuclear viral inclusions requires the early region 4 34-kilodalton protein.

The distribution of the adenovirus early region 1B 55-kDa protein (E1B-55kDa) in lytically infected HeLa cells was determined. At the time of infection, when the E1B-55kDa protein facilitates the cytoplasmic accumulation of viral mRNA while simultaneously restricting the accumulation of most cellular mRNA, five distinct intracellular localizations of the protein were observed. Only one of these was disrupted when cells were infected with a mutant virus that fails to produce a second viral protein encoded by early region 4 (E4-34kDa). This protein normally forms a complex with the E1B-55kDa polypeptide, enabling it to influence RNA metabolism. This key localization of the E1B protein was within and about the periphery of nuclear viral inclusion bodies believed to be the site of viral DNA replication and transcription. In the absence of the E4-34kDa protein, the coincidence of E1B-55kDa-specific immunofluorescence and phase-dense viral inclusions was reduced compared with that in a wild-type infection. Similarly, by immunoelectron microscopy, the relative number of E1B-55kDa-specific immunogold particles associated with the clear fibrillar inclusion bodies was reduced. However, the E4-34kDa protein was not required for the close association of the early region 2A DNA binding protein with the viral inclusions. We propose that the viral 55-kDa-34-kDa protein complex interacts with a cellular factor required for cytoplasmic accumulation of mRNAs and directs it to the periphery of the transcriptionally active viral inclusion bodies. This model provides an explanation for the ability of these viral proteins to simultaneously enhance accumulation of viral mRNAs and inhibit accumulation of cellular mRNAs.

Adenovirus Early Proteins↗
PubMed1980

Comparative effects of different strains of Corynebacterium parvum on natural cell-mediated cytotoxicity.

Because there are conflicting reports regarding the effects of Corynebacterium parvum (CP) on natural killer (NK) cell activity, several different strains of CP were compared. In replicate experiments, age- and sex-matched mice received 0.25-mg i.p. injections of one of four types of CP; formalin-killed strain 6134; heat-killed strain 6134; formalin-killed strain 5888 (actually Corynebacterium granulosum); or formalin-killed CP from the Pasteur Institute. At various days thereafter, two to three mice from each group were sacrificed to determine spleen weight, cellularity, and NK cell activity versus YAC-1 lymphoma cells. The CP from the Pasteur Institute augmented NK cell activity 3 days following injections; however, the activity returned to normal by Day 7 and remained at that level. On the other hand, strain 5888 did not cause as great an increase in lytic activity as did the Pasteur Institute CP at Day 3; but by Day 10 after injection, NK cell levels were above control, and they remained elevated through Day 21. Both the heat-killed and formalin-killed preparations of strain 6134 stimulated NK cell activity initially but resulted in a loss of activity at the later times tested. Experiments done with different doses and routes of injection yielded similar results. Thus, we were able to demonstrate that different types of CP have different effects on NK cell activity in mice and that the general kinetics of these effects were independent of dose or route of administration.

Animals↗
PubMed2000

Microbiological and molecular impacts of AbiK on the lytic cycle of Lactococcus lactis phages of the 936 and P335 species.

The lactococcal abortive infection mechanism AbiK was previously shown to be highly effective against the small isometric-headed bacteriophage ul36 of the P335 species, as evidenced by an efficiency of plaquing (e.o.p.) of 10(-6), a 14-fold reduction in the burst size and an efficiency at which centres of infection form (e.c.o.i.) of 0.5%. No phage DNA was detected in the infected AbiK+ cells [Emond, E., Holler, B. J., Boucher, I., Vandenbergh, P. A., Vedamuthu, E. R., Kondo, J. K. & Moineau, S. (1997). Appl Environ Microbiol 63, 1274-1283]. Here, the effects of AbiK are compared on the small isometric-headed phages p2 and P008 (936 species) and on the phage P335 (P335 species). The microbiological impacts of AbiK on p2 were relatively similar to those reported for ul36, with an e.o.p. of 10(6), an 11-fold reduction in the burst size and an e.c.o.i. of 5%. Contrary to phage ul36, replication of phage p2 DNA was observed in the AbiK+ cells. Only immature forms (concatemeric and circular DNA) of phage p2 DNA were found, indicating that the presence of AbiK prevented phage DNA maturation. These distinct molecular consequences of AbiK were also observed for phages P335 and P008, two phages that propagate on the same host. To the knowledge of the authors, this is the first time that different phage responses towards an Abi system have been reported.

Bacterial Proteins↗
PubMed1995

The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis.

Lytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from he cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effect on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated teh expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.

Animals↗