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[Isolation and characterization of "Listeria monocytogenes" bacteriophages (author's transl)].

Fifteen L. monocytogenes phages are isolated from lysogenic strains without induction and are selected for phage-typing. Four of these phages are produced by strains belonging to serotype 1/2a and 1/2b, eight by strains 4b and three other phages respectively by strains 4ab, 4g and 3c. Electron micrographs of 5 of these phages show isometric heads with non contractile tails. They belong to morphological group B of Bradley. Rabbits are immunized with 7 bacteriophages and all the phages are studied by neutralization tests with these 7 antisera. This study seems complex nevertheless it is possible to distinguish four antigenic groups: A, B, C, and D. The lytic spectrum of each phage is usually restricted to strains of the serotype from which that phage is obtained. It appears that a good correlation exists between phage sensitivity and serological type of the strains. With this first set of fifteen phages, 159 of 214 (74,3%) strains of L. monocytogenes can be classified in more than 21 types but it appears that some new phages obtained from strains of serotype 1/2 must be isolated in the future. Our data indicate that these phages can be used as a phage-typing scheme for L. monocytogenes.

Antigens, Viral↗
PubMed1977

The tumor enhancing property of herpes simplex virus type-2 (HSV-2).

The s.c. infection of DBF-1 mice with HSV-2 has a tumor enhancing effect on simultaneously i.m. implanted MCA-induced syngeneic spindle cell sarcoma. Thus with the dosage used here, the first palpable tumors appeared on day 6 in the virus treated group as compared to day 9 p.i. in the sham treated batch. Further, more tumors were formed: 75% among the infected and 40% among the sham injected. The tumor yield could be modulated: no difference between the 2 groups was found when the tumor cell dose was increased sufficiently to advance the first appearance of tumors to less than 6 days. The same result was obtained when the neoplastic cells were implanted 3 days ahead of injection of the virus. When the dose of the cells was decreased, no tumors were found in the sham treated batch, whereas there were some in the infected group. Previous in vitro mixing of the neoplastic cells with virus completely prevented tumor formation, probably due to their lysis by the virus. In fact, in tissue cultures, MCA cells become lytically infected with HSV-2. It is speculated that a similar situation occurs in acutely ill mice which die before the 13th day p.i., i.e., the tumor cells are destroyed by the virus. In fact, those animals, as a rule are free of neoplasmas. Thus it appears that an acute HSV-2 infection prevents MCA sarcoma whereas a latent infection promotes its growth. The possible reasons for this tumor enhancement caused by HSV-2 infection are discussed.

Animals↗
PubMed2005

JC virus induces altered patterns of cellular gene expression: interferon-inducible genes as major transcriptional targets.

Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

Cell Line↗
PubMed2004

Human CD8+ T cells recognize the 60-kDa cysteine-rich outer membrane protein from Chlamydia trachomatis.

The intracellular bacterial pathogen Chlamydia is sequestered from the host cell cytoplasm by remaining within an inclusion body during its replication cycle. Nevertheless, CD8(+) T cells recognizing Chlamydia Ags in the context of MHC class I molecules are primed during infection. We have recently described derivation of Chlamydia-specific human CD8(+) T cells by using infected dendritic cells as a surrogate system to reflect Chlamydia-specific CD8(+) T cell responses in vivo. These CD8(+) T cell clones recognize chlamydial Ags processed via the conventional class Ia processing pathway, as assessed by treatment of infected APC with lactacystin and brefeldin A, suggesting that the Ags are translocated from the chlamydial inclusion into the host cell cytosol. In this study, outer membrane protein 2 (OmcB) was identified as the Ag recognized by one of these Chlamydia-specific human CD8(+) T cells, and we defined the HLA*A0101-restricted epitope from this Ag. CD8(+) T cell responses to this epitope were present at high frequencies in the peripheral blood of both of two HLA*A0101 donors tested. In vitro chlamydial growth was completely inhibited by the OmcB-specific CD8(+) T cell clone independently of lytic mechanisms. OmcB is a 60-kDa protein that has been postulated to be associated with the Chlamydia outer membrane complex. The subcellular localization of OmcB to the cytosol of infected cells, as determined by conventional MHC class I Ag processing and presentation, suggests the possibility of an additional, cytosolic-associated function for this protein.

Adolescent↗
PubMed2000

Gene therapy for prostate cancer: where are we now?

PURPOSE: The ability to recombine specifically and alter DNA sequences followed by techniques to transfer these sequences or even whole genes into normal and diseased cells has revolutionized medical research and ushered the clinicians of today into the age of gene therapy. We provide urologists a review of relevant background information, outline current treatment strategies and clinical trials, and delineate current challenges facing the field of gene therapy for advanced prostate cancer. MATERIALS AND METHODS: We comprehensively reviewed the literature, including PubMed and recent abstract proceedings from national meetings, relevant to gene therapy and advanced prostate cancer. We selected for review literature representative of the principal scientific background for current gene therapy strategies and National Institutes of Health Recombinant DNA Advisory Committee approved clinical trials. RESULTS: Current prostate cancer gene therapy strategies include correcting aberrant gene expression, exploiting programmed cell death pathways, targeting critical cell biological functions, introducing toxic or cell lytic suicide genes, enhancing the immune system antitumor response and combining treatment with conventional cytotoxic chemotherapy or radiation therapy. CONCLUSIONS: Many challenges lie ahead for gene therapy, including improving DNA transfer efficiency to cells locally and at distant sites, enhancing levels of gene expression and overcoming immune responses that limit the time that genes are expressed. Nevertheless, despite these current challenges it is almost certain that gene therapy will be part of the urological armamentarium against prostate cancer in this century.

Adenoviridae↗
PubMed1976

Specialized transduction of colicin E1 DNA in Escherichia coli K-12.

Genetic studies were made on E. coli K-12 TM96, which carries recombinant molecules constructed by in vitro combination of colicin E1 DNA and a DNA fragment of E. coli for guanine synthesis derived from transducing phage. The recombinant molecules existed as stable plasmids within the cell and contained genes for colicin E1 immunity and the guaA enzyme (xanthosine 5'-monophosphate aminase) together with a part of the lambda genome, R through J: (R-A-F-J)+. A block of the lambda genome, int through Q, was not detected in the recombinant molecule. Thus, this recombinant molecule was named ColEl-coslambda-guaA, and the specialized tranduction of the ColEl-coslambda-guA DNA into various E. coli K-12 cells by lambda phage was described. Lysates prepared by lytic infection of lambda phage onto TM96 or by induction of TM96(lambda) lysogens contained transducing particles which could transduce gua-deleted E. coli to stable guaA+ cells. These transductants were proved to have similar genetic properties as those of TM96. The frequency of transduction was not affected by the presence of an attachement site for lambda, prophage lambda, colicin E1 plasmids, or the recA property within gua-deleted recipient cells. Transducing particles were resistant to EDTA treatment and most of them had an average density of about 1.472. This value corresponds to that of lambda phage particles, which contain about 72% of the lenght of lambda DNA.

Colicins↗
PubMed1978

Characterisation of mycobacteriophage ATCC 11759. Unusual physiocochemical properties of its DNA.

Growth conditions and a purification procedure for mycobacteriophage ATCC 11759, a lytic phage for Mycobacterium smegmatis, are described. The phage is a large DNA phage with a very long tail (240 nm). A study of its DNA revealed three interesting features. 1. After denaturation the DNA molecule yields two strands of different buoyant densities. 2. The native DNA has unusual physical properties: its buoyant density in CsC1 is very low (1.654 g/cm3), its sedimentation rate is lower than expected for the molecular weight, its thermal stability at low ionic strength is high. 3. The DNA (in its native form or after reannealing) is resistant to various restriction endonucleases.

DNA Restriction Enzymes↗
PubMed1972

Deoxyribonucleic acid synthesis in bacteriophage SPO1-infected Bacillus subtilis. I. Bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerase-deficient strains.

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol(-)) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg(2+) ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol(-) mutation had no effect on the synthesis of phage DNA or production of mature phage particles.

Adenosine Triphosphate↗
PubMed1987

Evolutionary relationship between plasmids and phages: phasmid P4 as a model.

Phasmid (phage-plasmid) P4 is capable of propagating as a temperate phage, lytic helper-dependent phage, and as a plasmid (pP4vir1). Alteration of cellular growth conditions can be used to manipulate the copy number (n) of pP4vir1 from low (n = 1-2) to high (n = 30-40). The levels of gene expression of P4 in the plasmid and phage states are radically different. While there is high expression of P4 proteins in the phage state, low levels of gene expression are found in the plasmid mode of propagation thereby allowing pP4vir1 to remain intracellularly without perturbing host homeostasis. Possible evolutionary origins of phasmid P4 are discussed.

Bacteriophage P2↗
PubMed1977

Genetic and physiological control of host cell lysis by bacteriophage lambda.

The timing of host cell lysis at the end of the lytic cycle of phage lambda is under complex control. The lambda S protein stimulates lysis. Another physiological system, the lysis regulator, inhibitis lysis from occurring prematurely. The effects of a series of phage and bacterial mutations on these controls are described. They show that the lambda rex gene plays a role in regulating lysis under suboptimal growth conditions. In certain mutant cells, and especially under anaerobic culture conditions, the rex gene aids in the scheduling of host cell lysis. The data also suggest that the lysis regulator may control the transition of the lambda S protein from an inactive to an active state.

Anaerobiosis↗
PubMed1983

Enzyme-linked immunosorbent assay for the detection of Epstein-Barr virus-induced antigens and antibodies.

Two Epstein-Barr virus (EBV)-specific ELISA tests were developed. One, based on the use of crude extracts from virus producer cells highly induced in the presence of Ara C (providing EA + VCA- cells) or in the absence of the drug (providing EA + VCA + cells) is suitable for the detection of antibodies directed against antigen complexes associated with the lytic virus cycle; i.e., EA, VCA and presumably also MA. The second, performed with purified EBNA, can be used for the detection of antibodies to the transformation-associated nuclear antigen. The tests are expected to find application in the dissection of antibody responses of patients to various antigenic subcomponents, the monitoring of EBV-coded antigens during biochemical purification, and the screening of spent media from hybridoma cultures for EBV-specific antibodies.

Antibodies, Viral↗
PubMed2005

HSV-1-derived recombinant and amplicon vectors for gene transfer and gene therapy.

Herpes simplex virus type 1 (HSV-1) is a major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections. Although in most cell types infection with HSV-1 will induce toxic effects ending in the death of the infected cells, the very deep knowledge we possess on the genetics and molecular biology of HSV-1 has permitted the deletion of most toxic genes and the development of non-pathogenic HSV-1-based vectors for gene transfer. Several unique features of HSV-1 make vectors derived from this virus very appealing for preventive or therapeutic gene transfer. These include (i) the very high transgenic capacity of the virus particle, authorizing to convey very large pieces of foreign DNA to the nucleus of mammalian cells, (ii) the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and (iii) the ability of HSV-1 vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia and probably in other neurons as well, from where transgenes can be strongly and long-term expressed. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors, and defective helper-dependent vectors known as amplicons. Each of these different vectors attempts to exploit one or more of the above-mentioned features of HSV-1. In this review we will update the current know-how concerning design, construction, and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

Gene Transfer Techniques↗
PubMed2007

Structure and function of latency-associated nuclear antigen.

Latency-associated nuclear antigen (LANA) encoded by open reading frame 73 (ORF73) is the major latent protein expressed in all forms of KSHV-associated malignancies. LANA is a large (222-234 kDa) nuclear protein that interacts with various cellular as well as viral proteins. LANA has been classified as an oncogenic protein as it dysregulates various cellular pathways including tumor suppressor pathways associated with pRb and p53 and can transform primary rat embryo fibroblasts in cooperation with the cellular oncogene Hras. It associates with GSK-3beta, an important modulator of Wnt signaling pathway leading to the accumulation of cytoplasmic beta-catenin, which upregulates Tcf/Lef regulated genes after entering into the nucleus. LANA also blocks the expression of RTA, the reactivation transcriptional activator, which is critical for the latency to lytic switch, and thus helps in maintaining viral latency. LANA tethers the viral episomal DNA to the host chromosomes by directly binding to its cognate binding sequence within the TR region of the genome through its C terminus and to the nucleosomes through the N terminus of the molecule. Tethering to the host chromosomes helps in efficient partitioning of the viral episomes in the dividing cells. Disruptions of LANA expression led to reduction in the episomal copies of the viral DNA, supporting its role in persistence of the viral DNA. The functions known so far suggest that LANA is a key player in KSHV-mediated pathogenesis.

Animals↗
PubMed1984

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication.

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including phi18. The efficiency of plating of phi18 on ME2 and N1 could be increased from <1 x 10 to 5.0 x 10 and from 7.6 x 10 to 2.1 x 10, respectively, when the host strains were subcultured at 40 degrees C before plating the phage and the phage assay plates were incubated at 40 degrees C. Host-dependent replication was demonstrated in N1 at 30 degrees C and in N1 and ME2 at 40 degrees C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to phi18 at 40 degrees C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40 degrees C showed an increased ability to adsorb phi18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a phi18 preparation that had been previously modified for growth on N1 indicated that at 40 degrees C phage development was characterized by a shorter latent period and larger burst size than at 30 degrees C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30 degrees C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.

Journal Article↗
PubMed1981

Genetic complementation by cloned bacteriophage T4 late genes.

Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome.

Bacteriophage lambda↗
PubMed1997

Repression of host RNA polymerase II transcription by herpes simplex virus type 1.

Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II.

Cell Line↗
PubMed1986

Role of the adenovirus early region 1B tumor antigens in transformation and lytic infection.

We have investigated the contribution of each of the two adenovirus type 5 (Ad5) major early region 1b (E1b) proteins in cell transformation and in lytic infection. An Ad5 E1 plasmid, in which the reading frame for the 19-kDa E1b protein was abolished by a stop codon close to the initiation codon, transformed primary baby rat kidney (BRK) cells with an efficiency of about half of that of a wild type Ad5 E1 plasmid, whereas a plasmid with a mutation in the gene for the 58-kDa E1b protein transformed the same primary cells with only one-third of the wild type efficiency. Plasmids containing region E1a only or a plasmid carrying mutations in the genes for major E1b proteins all transformed primary cells with an efficiency of approximately 5% of wild type. To test the effect of the E1b mutations in virion-mediated cell transformation, the mutant E1b regions were introduced into intact viral genomes by overlap recombination and were subsequently used in a transformation assay on BRK cells. The 19 and 58-kDa mutant viruses were found to transform BRK cells with 11 and 25% of the efficiency of wild type virus, respectively. These results suggest that the 19-kDa E1b protein is essential for virus-mediated cell transformation, in agreement with results of others, but not for plasmid-mediated cell transformation. In lytic infection, the 19-kDa mutant virus was some 30-fold reduced in yield on HeLa cells, whereas the 58-kDa mutant virus was 3000-fold reduced in its ability to grow on HeLa cells at low multiplicity of infection, but showed a marked multiplicity-dependent leakiness. The 58-kDa mutant virus was not defective when its growth was assayed on human embryonic kidney (HEK) cells. This may indicate that cellular proteins are expressed in HEK cells that are functionally homologous to the 58-kDa E1b protein.

Adenovirus Early Proteins↗
PubMed1990

Quantitative in situ hybridization using strand specific RNA probes: expression of the bunyavirus Germiston S segment in mosquito cells.

Infection of Vero (monkey) cells by Germiston bunyavirus is highly cytopathic with cell lysis and virus production at a high titre, whereas infection of Aedes albopictus C6/36 (mosquito) cells leads, after an acute primary phase, to a persistent non-cytopathic infection with a loss in virus production. In this report we demonstrate that single-stranded RNA probes can be successfully used in an in situ hybridization assay to quantify viral expression during this persistent infection. The steady-state levels of viral S-RNA segment (genomic and messenger sense) during the acute phase were similar to those observed in lytically infected Vero cells, but appeared delayed. Both senses of S-RNA were detected throughout persistent infection but in lower amounts, in less than 10% of the cells and always in the cytoplasm of infected cells. The number of copies per cell of messenger sense S-RNAs remained low during persistent infection whereas a higher fluctuation was observed for genomic S-RNAs. In situ hybridization with specific stranded RNA probes provides both qualitative and quantitative informations, that can lead to a better understanding of virus-cell interactions.

Aedes↗