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PubMed2005

Increased efficiency of phorbol ester-induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus during S phase.

Expression of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic genes is thought to be essential for the establishment and progression of KSHV-induced diseases. The inefficiency of lytic reactivation in various in vitro systems hampers the study of lytic genes in the context of whole virus. We report here increased expression of KSHV lytic genes and increased release of progeny virus when synchronized cultures of body cavity-based lymphoma-1 cells are treated with a phorbol ester during S phase of the cell cycle.

DNA Replication↗

PubMed1972

Acquisition of sequences homologous to host deoxyribonucleic acid by closed circular simian virus 40 deoxyribonucleic acid.

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.

Animals↗

PubMed1985

Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1).

Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.

Animals↗

PubMed1992

Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector.

Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system. We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3. This vector, denoted Cgal delta 3, contains the E. coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome. We studied the efficiency of Cgal delta 3 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer. Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS. Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia. Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expression of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression. Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.

Animals↗

PubMed1996

Cytopathology of Bay of Piran shrimp virus (BPSV), a new crustacean virus from the Mediterranean Sea.

A new picorna-like or parvo-like virus was found in the hepatopancreas of the shrimp Palaemon elegans from the Bay of Piran (Mediterranean Sea). Bay of Piran shrimp virus (BPSV) infects preferably the R-cells of the hepatopancreas and to a lesser extent the F-cells. In R-cells it is abundant in the cytoplasm and within membrane whorls. In lesser numbers it can also be present in cell organelles like the endoplasmic reticulum, autophagosomes, or mitochondria. Less contrasting virus-like particles are observed in the nucleoplasm, suggesting a nuclear replication of the virus and completion of assembly in the cytoplasm. In F-cells the virus is located mostly in cisternae of the Golgi bodies. Occasionally, there are also virus clusters in the cytoplasm. B-cells are only rarely infected. The virus seems to invade the host cells from the lumen of the hepatopancreatic tubules through the microvillous border. Mature viruses are released again into the hepatopancreatic lumen by discharge of lytic epithelial cells. Individual viruses can also penetrate through the basal cell membrane into the hemal space. Occasionally, fixed phagocytes are attached to the basal lamina of heavily infected hepatopancreas cells, indicating an activation of the immune defense system by the virus.

Animals↗

PubMed1988

Growth pattern of various JHM coronavirus isolates in primary rat glial cell cultures correlates with differing neurotropism in vivo.

The JHM strain of murine hepatitis coronavirus is neurotropic in rats, causing either fatal acute encephalomyelitis or subacute demyelinating encephalomyelitis. We have examined the growth properties of three JHM virus isolates in primary rat glial cultures and found a correlation with their ability to cause disease. Wild type JHM virus has the propensity to cause lytic infections in glial cultures, and a temperature-sensitive mutant designated JHM-ts43 invariably produces persistent infections with reduced cytopathic effects (CPE) as compared to the wild type. Moreover, a non-neurotropic isolate, designated JHM-Pi virus, produces either non-productive persistent infections at low multiplicity of infection (m.o.i.) or productive persistent infections at high m.o.i., with, however, no CPE. The phenotypic expression of persistence is glial cell-dependent, since all three viruses produce similarly lytic infections when grown on various susceptible cell lines. The genetic basis of JHM virus persistence can be explained at the level of direct virus-glial cell interactions.

Animals↗

PubMed2001

Control of Epstein-Barr virus reactivation by activated CD40 and viral latent membrane protein 1.

In humans, Epstein-Barr virus (EBV) establishes a persistent latent infection in peripheral resting B lymphocytes. Virus reactivation is highly restricted. Whereas in healthy humans the infection usually is benign, immunocompromised patients show an increased risk for EBV-associated malignancies, accompanied by an increase in virus replication and in the number of virus-infected cells. To search for viral and host factors regulating virus reactivation, we used conditionally EBV-immortalized B cells. We found that CD40-CD40 ligand interaction and the viral mimic of activated CD40, EBV latent membrane protein 1, suppress virus reactivation. Both inhibit anti-IgM or phorbolester-induced transcription of the viral immediate early protein BZLF1, which controls entry into the viral lytic cycle. The finding that latent membrane protein 1 and CD40 contribute to the regulation of latency may have important implications for the balance between EBV and its host in normal as well as in immunocompromised individuals.

B-Lymphocytes↗

PubMed1992

On the kinetics and optimal specificity of cytotoxic reactions mediated by T-lymphocyte clones.

Using the chromium release assay and the single cell assay in agarose, we study the cytotoxic reaction of the MHC-restricted T lymphocyte clones P89:15 and P1:3, which recognize distinct but specific tumour antigens on the surface of syngeneic P815 mastocytoma cells. We propose a mathematical model which describes these experiments, accounts for the strongly non-Michaelian behaviour of the reaction and permits us to estimate the kinetic parameters characterizing effector-target conjugation and lethal hit delivery. The results show that the binding and lytic activity of effector cells is modulated by the number of targets bound to them. The binding of a second target by an effector having already a target bound is facilitated; on the other hand, an effector having bound two targets delivers a lethal hit more slowly than one with a single target bound. We investigate the role of these kinetic properties in the competition between the process of tumour progression due to cancer cell replication and the process of tumour regression due to T lymphocyte cytotoxic activity. For both clones, we estimate the effector-target ratio beyond which rejection prevails. This ratio is nine times larger for P1:3 than for P89:15. Furthermore, our analysis suggests that there exists an optimal specificity minimizing this ratio. Deviations from this optimum, be it in the sense of an increase or decrease of specificity, tends to stabilize the tumoural state: a situation which in the broader context of the immune response evolution and regulation can be viewed as an immune response dilemma.

Animals↗

PubMed2000

Complete nucleotide sequence, molecular analysis and genome structure of bacteriophage A118 of Listeria monocytogenes: implications for phage evolution.

A118 is a temperate phage isolated from Listeria monocytogenes. In this study, we report the entire nucleotide sequence and structural analysis of its 40 834 bp DNA. Electron microscopic and enzymatic analyses revealed that the A118 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules. No evidence for cohesive ends or for a terminase recognition (pac) site could be obtained, suggesting that A118 viral DNA is packaged via a headful mechanism. Partial denaturation mapping of DNA cross-linked to the tail shaft indicated that DNA packaging proceeds from left to right with respect to the arbitrary genomic map and the direction of genes necessary for lytic development. Seventy-two open reading frames (ORFs) were identified on the A118 genome, which are apparently organized in a life cycle-specific manner into at least three major transcriptional units. N-terminal amino acid sequencing, bioinformatic analyses and functional characterizations enabled the assignment of possible functions to 26 ORFs, which included DNA packaging proteins, morphopoetic proteins, lysis components, lysogeny control-associated functions and proteins necessary for DNA recombination, modification and replication. Comparative analysis of the A118 genome structure with other bacteriophages revealed local, but sometimes extensive, similarities to a number of phages spanning a broader phylogenetic range of various low G+C host bacteria, which implies relatively recent exchange of genes or genetic modules. We have also identified the A118 attachment site attP and the corresponding attB in Listeria monocytogenes, and show that site-specific integration of the A118 prophage by the A118 integrase occurs into a host gene homologous to comK of Bacillus subtilis, an autoregulatory gene specifying the major competence transcription factor.

Amino Acid Sequence↗

PubMed1999

Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new transcript, initiated at an unidentified site 5' of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.

B-Lymphocytes↗

PubMed1979

Structural and biological properties of mycoplasmavirus MVL3: an unusual virus-procaryote interaction.

The kinetics of adsorption and growth of mycoplasmavirus MVL3 in Acholeplasma laidlawii 1305/68 host cells have been studied with one-step growth, premature lysis, and single-burst experiments. The virus was found to kill infected host cells. Virus release starts 90 min after infection and continues for about 10 to 15 h. Hence, virus production is unlike the classical lytic bacteriophages and instead resembles nonlytic cytocidal animal viruses. Structural details of the virus are described, and the molecular weight of the viral linear DNA has beenfound to be 26 x 10(6).

Acholeplasma laidlawii↗

PubMed1998

Characterization of defective viral RNA produced during persistent infection of Vero cells with Murray Valley encephalitis virus.

Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.

Animals↗

PubMed2005

Prevention of relapse after chemotherapy in a chronic intracellular infection: mechanisms in experimental visceral leishmaniasis.

In visceral leishmaniasis, chemotherapy probably seldom eradicates all parasites in tissue macrophages; nevertheless, most T cell-intact patients show long-lasting clinical cure after treatment despite residual intracellular infection. To characterize prevention of posttreatment relapse, amphotericin B was used to kill approximately 90-95% of Leishmania donovani in livers of mice deficient in mechanisms of acquired antileishmanial resistance. Recrudescence subsequently developed 1) in animals deficient in both CD4 and CD8 T cells as well as CD40L-mediated T cell costimulation, but not in a) CD4 or CD8 cells alone, b) NK cell lytic activity, or c) ICAM-1-recruited monocytes; and 2) in mice deficient in IFN-gamma, but not in the IFN-gamma-inducing cytokines, a) IL-12, b) IL-12 and IL-23, or c) IL-18. Posttreatment recrudescence also did not develop in animals deficient in macrophage phagocyte NADPH oxidase (phox) or inducible NO synthase (iNOS) alone or, surprisingly, in those deficient in both phox and iNOS. Therefore, regulation of the intracellular replication of residual Leishmania donovani that escape chemotherapy evolves to a host mechanism distinguishable from initial acquired resistance at the T cell, cytokine, and macrophage levels. Posttreatment, either CD8 or CD4 cells can direct the response, IL-12 is not required, and iNOS and phox, the activated macrophage's primary IFN-gamma-inducible leishmanicidal pathways, both become dispensable.

Amphotericin B↗

PubMed1995

[Origin and properties of Pseudomonas aeruginosa PAO1 clones surviving after induction of prophages-transposons].

Various mutations cancelling the lethal effect of phage lytic development and simultaneous phenotypic modifications were found in rare clones surviving after incubation at 42 degrees C of Pseudomonas aeruginosa (D3112 cts 15), lysogenic for thermoinducible mutant cts 15 of the transposable prophage (TP) D3112. All mutations arose prior to thermal induction. Temperature induction of other bacteriophages (nontransposable) did not lead to selection of bacterial morphological mutants. Therefore, it was concluded that mutagenesis occurred upon the partial (reversible) TP derepression accompanied by coupled replication-transposition of TP, the latter being the direct cause of the mutator effect. Isolation of the P. aeruginosa PAO1 mutant R10 (this mutant is resistant to infection with TP at 42 degrees C) allowed the proper selection and examination of numerous survivors. Comparison of their types derived from lysogens with different prophage location indicated that the number of secondary sites where TP integration is possible without the loss of cell viability is limited. Several transposition events occurred in the history of some survivors (during a repeated or single derepression event). Type D clones, which produce small colonies, are of special interest, because mechanisms underlying the survival of such clones are extremely diverse, and their phenotypes indicate the possibility of stable chromosomal rearrangements in the genome of P. aeruginosa.

Cloning, Molecular↗

PubMed1981

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

Adenoviridae↗

PubMed1990

In vitro replication and cytopathogenicity of the feline immunodeficiency virus for feline T4 thymic lymphoma 3201 cells.

Cytotoxic feline immunodeficiency virus (FIV) infection was established in feline T4 thymic lymphoma 3201 cells with the Petaluma isolate of the feline immunodeficiency virus (FIV-Petaluma). Mg2(+)-dependent, reverse transcriptase (Mg2+ RT) activity and FIV p24/28-positive cells were evident beginning at 18 days postinoculation (dpi). Cell death was observed beginning at 22 dpi, with a maximum of 40% dead (trypan blue dye exclusion at 26 dpi). This cytocidal change was not observed in cultured Crandell feline kidney fibroblasts similarly infected with FIV-Petaluma. The surviving cells grew out and a chronic FIV-producer cell line was established. The 3201 cell-derived FIV (FIV-3201) was far more virulent for FIV-naive feline 3201 cells, with FIV p24/28-positive cells and Mg2+ RT activity first detectable by 4-8 dpi and subsequent loss of cell viability detectable by 8-12 dpi. Maximum kill (40% dead) was observed at 16 dpi. Comparison between viral infectivity of FIV-Petaluma and FIV-3201 for FIV-naive 3201 cells showed an increase of 1 log10 tissue culture infectious doses (TCID50) by amplification/passage in 3201 cells. Cytologic and electron microscopic examination of 3201 cells in FIV-infected cultures showed frequent budding lentiviral particles. This lytic infection system opens the way to the routine detection, isolation, and quantitation of FIV from FIV-infected cats, to the large-scale propagation of the virus, and to a system for evaluation of the mechanisms of FIV lymphocytotoxicity and the development of therapies to counteract lentiviral cytopathicity.

Animals↗

PubMed2001

Intraneoplastic polymer-based delivery of cyclophosphamide for intratumoral bioconversion by a replicating oncolytic viral vector.

rRp450 is an oncolytic herpesvirus that expresses the CYP2B1 cDNA, responsible for bioconverting cyclophosphamide (CPA) into the active metabolites 4-hydroxyCPA/aldophosphamide (AP). However, formal proof of prodrug activation is lacking. We report that activation of CPA in cells infected with rRp450 generates a time-dependent increase of diffusible 4-hydroxyCPA/AP. For in vivo applications, a CPA-impregnated polymer was implanted into human tumor xenografts inoculated with rRp450. The area under the curve for 4-hydroxyCPA/AP was 806 microg/g of tumor tissue/h when CPA was administered via intraneoplastic polymer and 3 microg/g of tumor tissue/h when CPA was administered i.p. Therefore, (a) a lytic virus expressing a "suicide" gene can activate a prodrug; and (b) within rRp450-infected tumor, more prolonged and higher concentrations of activated metabolites are generated by intraneoplastic compared with systemic administration of prodrug.

Animals↗

PubMed2004

Future directions for the field of oncolytic virotherapy: a perspective on the use of vaccinia virus.

Oncolytic virotherapy is an emerging biotherapeutic platform based on genetic engineering of viruses capable of selectively infecting and replicating within cancer cells. Such viruses have been found to be both safe and to produce antitumour effects in a number of Phase I and II clinical trials. Early work in this field has been pioneered with strains of adenovirus which, although well suited to gene therapy approaches, have displayed certain limitations in their ability to directly destroy and spread through tumour tissues, particularly after systemic administration. Investigators have subsequently been examining the feasibility of using a variety of different viruses as oncolytic agents. Vaccinia virus is perhaps the most widely administered and successful medical product in history; it displays many of the qualities thought necessary for an effective antitumour agent and is particularly well characterised in people due to its role in the eradication of smallpox. Vaccinia has a short life cycle and rapid spread, strong lytic ability, inherent systemic tumour targeting, a large cloning capacity and well-defined molecular biology. In addition, the virus produces no known disease in humans, has been delivered safely to millions of people and has already demonstrated antitumoural efficacy in trials with vaccine strains. These qualities, along with strategies for further improving the safety and antitumour effectiveness of vaccinia, will be discussed in relation to the broad spectrum of clinical experience already achieved with this virus in cancer therapy.

Antigens, Neoplasm↗