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PubMed1981

Degradation of intracellular DNA in KB cells infected with cyt mutants of human adenovirus type 12.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.

Adenoviruses, Human↗

PubMed1995

Detection and intracellular localization of equine herpesvirus 1 IR1 and IR2 gene products by using monoclonal antibodies.

During lytic infection, two transcripts arise from the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene (IR1): a single, spliced 6.0-kb IE mRNA and a 3'-coterminal 4.4-kb early mRNA (IR2). Previous studies demonstrated that transiently expressed IR1 and IR2 gene products are potent transcriptional regulators: IR1 proteins are capable of trans activating representative EHV-1 early and late promoters, while both IR1 proteins and the IR2 product, which lacks IR1 amino acid residues 1 to 322, trans repress the IR1 promoter. In the present study, monoclonal antibodies (MAbs) against the major IE protein, IE1, were developed, characterized as to their ability to detect IR1 and IR2 products, and used to examine extracellular virions for the presence of IE1-related proteins and to define the IR1 and IR2 protein synthesis and intracellular distribution in EHV-1-infected cells. The results demonstrated that (i) anti-IE1 MAbs representing three noncompetitive epitope-binding groups reacted with multiple IE protein species, as well as with a 146-kDa early protein identified as the putative IR2 gene product; (ii) the three reactive epitopes mapped to a region spanning amino acids 323 to 552 of IR1; (iii) anti-IE1 MAbs reacted with the 144-kDa in vitro-translated IR2 product and with a transiently expressed IR2 product similar in size; (iv) small amounts of IE1 and the 146-kDa protein were associated with the nucleocapsid-tegument fraction of mature virions; (v) in immunofluorescence assays of lytically infected cells, IR1-IR2 gene products were first detectable between 1 and 2 h postinfection as discrete, punctate, intranuclear foci; (vi) as the infection progressed, the intranuclear reactivity increased and redistributed into large, intensely stained nuclear compartments which corresponded to the sites of active viral DNA synthesis; (vii) fibrillar, as well as more generalized cytoplasmic staining, first observed at about 5 h postinfection, increased throughout infection; and (viii) while viral DNA synthesis was required for the progressive intranuclear redistribution, the cytoplasmic accumulation of IR1-IR2 proteins occurred subsequent to early infection events.

Animals↗

PubMed2004

Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1.

The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1vir). In several E. coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF+ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF+ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.

Apoptosis↗

PubMed1985

Characterization of streptococcal bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected.

Bacteriophages↗

PubMed1986

Recent clinical isolates of cytomegalovirus suppress human cytomegalovirus-specific human leukocyte antigen-restricted cytotoxic T-lymphocyte activity.

Peripheral blood mononuclear cells harvested from healthy adults seropositive for human cytomegalovirus (HCMV) and cultured with laboratory strain AD-169 demonstrated human leukocyte antigen-restricted and HCMV-specific killing on target cells infected with either HCMV laboratory strain AD-169 or recent low-passage HCMV isolates. These results indicated that the determinants recognized by cytotoxic T lymphocytes (CTLs) are shared among different strains of HCMV. However, when low-passage isolates, rather than high-passage AD-169 virions, were used to stimulate CTL activity, the lytic response was significantly lower against all targets. Mixing of AD-169 and low-passage HCMV isolates induced low CTL activity. Collectively, the findings suggest that low-passage HCMV isolates have dual effects--antigenic stimulation and immunosuppression--whereas laboratory strain AD-169 is primarily immunogenic. The study of several recent isolates indicated that they varied in their ratio of immunostimulation to suppression, that infectious virus was necessary to produce suppression, and that suppressive isolates did not have to be present at the initiation of culture to exert their suppressive effects.

Cytomegalovirus↗

PubMed1998

The relative contribution of antibody production and CD8+ T cell function to immune control of Trypanosoma cruzi.

The life cycle of the protozoan parasite Trypanosoma cruzi in mammalian hosts includes both non-dividing trypomastigote forms which circulate in the blood and replicating intracellular amastigotes which reside within the cytoplasm of a variety of host cells. In this study we have used mice with induced mutations in genes responsible for either antibody production or cytolytic T lymphocyte (CTL) function to examine the relative contributions of these effector mechanisms to control of T. cruzi. Mice deficient in the production of antibodies exhibited a delay in the rise in acute phase parasitaemia and an extended time to death relative to mice lacking CD8+ T cells. Nevertheless, B cell deficient mice eventually succumbed to the infection. Prior infection with an avirulent strain of T. cruzi failed to protect either CD8+ T cell-deficient mice or B cell deficient mice from challenge infection with virulent parasites. In contrast, mice with disruptions in the genes controlling perforin- or granzyme B-mediated cytolytic pathways had parasitaemia and mortality rates similar to wild-type mice and were protected from secondary infection by prior exposure to avirulent parasites. These results 1) confirm that antibody production, although secondary in importance to cellular responses, is nevertheless absolutely required and 2) perforin- or granzyme B-mediated lytic pathways are not required for control of T. cruzi infection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1983

Integration, loss, and reacquisition of defective viral DNA in SV40-transformed mouse cell lines.

We have examined the state of viral DNA in a set of SV40-transformed mouse cell lines. Using restriction enzymes which cut SV40 DNA in one place, we demonstrate that anchorage-independent SV40-transformed mouse cells commonly contain one or more detectable defective monomers of integrated viral DNA. The defective viral DNA in one of these cell lines, SV101, was extensively mapped using single and double enzyme digests. The results of this analysis indicate that SV101 contains nondefective viral DNA as well as defective viral DNA of the following sizes: 5.0, 4.3, 3.7, 3.4, and 1.5 kb. Three of these defective monomers (4.3, 3.7, and 1.5 kb) preserve the amino terminal exon of large T antigen, and two monomers (4.3, and 3.7 kb) preserve the little t coding region. Anchorage-dependent subclones of SV101 preferentially lose the defective viral DNA, while retaining an intact SV40 early region and the ability to express lytic-sized large and small T antigens. Despite a considerable amount of viral DNA rearrangement which accompanies subcloning, anchorage-independent subclones of SV101 retain defective viral DNA, especially the 4.3- and 3.7-kb monomers. Also, when an anchorage-independent subclone is selected from an anchorage-dependent revertant of SV101, it reacquires defective viral DNA, although of a size not seen in SV101. We conclude that defective viral DNA plays a role in generating the anchorage-independent phenotype. In earlier studies, we have reported that anchorage-transformed mouse lines contain a variant (100kDa) T antigen. The possible role of defective viral DNA in generating this T antigen is discussed.

Animals↗

PubMed1976

Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.

Adenoviridae↗

PubMed2001

CREB-binding protein and histone deacetylase regulate the transcriptional activity of Kaposi's sarcoma-associated herpesvirus open reading frame 50.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator, which binds to the KSHV promoter and stimulates the transcription of viral early and late genes, thus activating the lytic cycle of KSHV. We report here that KSHV ORF50 binds to the cellular proteins CREB-binding protein (CBP) and histone deacetylase (HDAC) and these binding events modulate ORF50-activated viral transcription. Binding of ORF50 to CBP and HDAC activates and represses, respectively, ORF50-mediated viral transcription. KSHV ORF50 was shown to bind to the C/H3 domain and the C-terminal transcriptional activation domain of CBP, while CBP bound to the amino-terminal basic domain and the carboxyl-terminal transactivation domain of ORF50. The LXXLL motif within the transcriptional activation domain of ORF50 is reminiscent of the CBP-binding sequence found in nuclear receptor proteins. The adenovirus E1A protein, which also binds to the C/H3 domain of CBP, repressed the transcriptional activation activity of ORF50. The cellular protein c-Jun, which binds to the kinase-induced activation domain of ORF50, stimulated ORF50-mediated viral transcription. The HDAC1-interacting domain of ORF50 was shown to be a central proline-rich sequence. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate its transcription and replication through interaction with both histone acetyltransferases and HDACs.

CREB-Binding Protein↗

PubMed1995

The feline lymphoid cell line MBM and its use for feline immunodeficiency virus isolation and quantitation.

We report on the development of a feline T lymphoblastoid cell line obtained from the peripheral blood mononuclear cells (PBMC) of a specific pathogen free cat and designated MBM. The cells are pan-T+, CD4- and CD8- and remained interleukin-2-dependent and concanavalin A-dependent throughout the period of observation. MBM cells have proved at least as sensitive as fresh blasts to infection with cell-free stocks of three feline immunodeficiency virus (FIV) isolates. Upon infection, they exhibit a lytic cytopathic effect. Repeated attempts to establish a chronic infection have failed. Using a limiting cell dilution method, it has been shown that MBM cells may be more sensitive than fresh blasts as substrate for isolating FIV from the PBMC of infected cats. These studies have also shown that considerable individual variations exist in the virus loads present in the PBMC of naturally infected cats, and that load size does not appear to correlate with cat age, clinical status, CD4/CD8 ratio and titer of serum neutralizing antibody.

Animals↗

PubMed1973

Human papovavirus, BK strain: biological studies including antigenic relationship to simian virus 40.

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.

Animals↗

PubMed1998

Molecular mechanisms of poliovirus persistence: key role of capsid determinants during the establishment phase.

As viral persistence is of major medical importance, well-characterized, simple models are needed to improve our understanding of persistent infections. We have chosen to study the molecular mechanisms of viral persistence with the poliovirus (PV), because this picornavirus is one of the best characterized animal viruses, it infects the central nervous system which is a target organ for viral persistence, and it belongs to the Picornaviridae family of viruses, which includes several naturally persisting viruses. We have developed models of PV persistence in neuronal and epidermoid cells, and the present review will focus on the latter one because both lytic and persistent PV strains can be used to study the PV-HEp-2 cell interactions. The viral determinants of persistence have been investigated with this model, and PV determinants have proven to be of crucial importance for the establishment of persistence in HEp-2 cells. Precise determinants of PV persistence have been identified for PV serotypes 1 and 3, in capsid proteins VP1 and VP2. These determinants modify the early steps of the PV cycle, and in particular, the conformational modifications of the capsid following virus adsorption onto its receptor. These results permit us to propose several hypotheses concerning PV persistence and the early steps of the PV cycle.

Animals↗

PubMed1993

Modification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0).

The outcome of herpes simplex virus type 1 (HSV-1) infection depends upon the interplay of both host and viral factors. During lytic infection, HSV-1 causes a loss of immunofluorescent staining of discrete nuclear domains (ND10). This elimination of the host's ND10 staining occurs under conditions that allow only HSV-1 immediate early viral gene expression. Western blot analysis indicates that the loss of ND10 staining is due to ND10 redistribution, rather than protein degradation or turnover. When deletion mutants of all of the HSV-1 immediate early genes were tested, only infection with an immediate early gene 1 product (ICP0) deletion mutant, d11403, was unable to eliminate ND10 antigen staining. Also, ICP0 transiently colocalized with ND10 antigens, after which ND10 antigens became undetectable. At late times during infection with d11403, the host ND10 antigens were retained in virus-induced structures which were never observed during wild-type HSV-1 infection. These results suggested that ICP0 may be directly involved in the modification of the host nuclear domain. Infection with an adenovirus recombinant that expressed ICP0 demonstrated that in the absence of other HSV-1 proteins ICP0 was sufficient for the change in nuclear distribution of host antigens located at ND10. We postulate that the trans-activation function of ICP0 during viral replication may be mediated by replacing, modifying or reorganizing nuclear host factors.

Animals↗

PubMed1995

Analysis of protein synthesis by two-dimensional gel electrophoresis in T cells persistently infected with coxsackie B virus.

Coxsackie B viruses (CBV) have been implicated in various human diseases that present as either limited acute infections or prolonged chronic infections. A number of investigations have suggested that a virus-induced immune dysfunction might play a role in in vivo pathogenesis. In the current study, we describe CBV infection of two human T cell-derived cell lines (Jurkat and MOLT-4 cells) as potential models for CBV infection of lymphocytes. Short term (up to 144 h post-infection) CBV infection of either cell line resulted in a decline in the viability of the cell population together with an approximate 10-fold rise in the titre of infectious virus during the period of incubation. Analyses of the intracellular proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) demonstrated that although putative virus proteins were detectable there was minimal inhibition of the cellular protein synthesis following CBV infection. This contrasted with the more permissive and highly lytic CBV infection of HEp-2C cells (Cash, Electrophoresis 1991, 10, 793-800). Persistently infected cell lines from both Jurkat and MOLT-4 cells (piJURKAT-3673 and piMOLT-2667 cells) were established. Analyses of intracellular protein synthesis of these persistently infected cell lines showed the synthesis of novel proteins not detected for the corresponding uninfected parental cell line. There were no significant alterations in overall cellular protein synthesis detectable by the small format 2-D PAGE system employed in these investigations. The data presented in the current investigation will contribute towards studies on virus-induced responses of specific biological functions associated with T cells.

Cell Line↗

PubMed1989

In vitro development of bone-marrow-derived macrophages. Influence of mouse genotype on response to colony-stimulating factors and autocrine interferon induction.

Responsiveness to granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) of bone marrow cells derived from different mouse strains was investigated. There were great variations in proliferation between different strains of inbred mice. Bone marrow cells from mouse strains with a high rate of proliferation in response to GM-CSF also had a high proliferating capacity to M-CSF. The response to either CSF did not correlate with a certain H-2 haplotype. GM-CSF induced consistently higher proliferation than M-CSF. Proliferation in response to M-CSF, but not to GM-CSF, could be enhanced by the addition of antibodies against interferon (IFN). IFN is the only known inducer of (2'-5') oligoadenylate (oligo (A] synthetase. This enzyme was induced in macrophages grown in the presence of M-CSF, but not in GM-CSF promoted cells. Enzyme induction was completely abrogated by simultaneous treatment with anti-IFN alpha/beta. Infection of macrophages with herpes simplex virus type 1 (HSV) and vesicular stomatitis virus (VSV) revealed that GM-CSF-promoted cells were highly susceptible to lytic infection by these viruses. In contrast, virus titres in M-CSF-cultured cells were 100-fold lower. We conclude that, contrary to M-CSF, GM-CSF does not induce autocrine IFN during haematopoiesis. As judged from data with BALB/c mice, the sensitivity to the anti-proliferative effect of the autocrine IFN may be a factor which influences M-CSF-promoted proliferation.

2',5'-Oligoadenylate Synthetase↗

PubMed1997

Infection of K562 cells with influenza A virus increases their susceptibility to natural killer lysis.

Natural killer (NK) cells play a role in the natural immunity against tumor cells. In the present study, we demonstrate that infection of the NK-sensitive tumor cell line K562 with influenza A virus caused a substantial increase in lysis of up to sevenfold when compared to noninfected cells. Similar to NK cells, IL-2-activated killer cells exhibited higher lytic activity against virus-infected K562 cells. This effect of the virus correlated with the increase in the expression of intracellular adhesion molecule-1 (ICAM-1) on K562 cells. Changes in the susceptibility to NK lysis were accompanied by alterations, within minutes, in the cytoskeleton as detected by intracellular fluorescein fluorescence polarization measured on the Cellscan, a static cytometer. The possible role of iCAM-1 and the cytoskeleton in the cytotoxic response of NK cells is discussed.

Cytotoxicity, Immunologic↗

PubMed1995

Effects of the DNA-damaging enediyne C-1027 on intracellular SV40 and genomic DNA in green monkey kidney BSC-1 cells.

This study describes the selective ability of C-1027 to induce limited double-strand damage in a viral DNA target. The effect of the cellular environment on C-1027 activity was examined by assaying the extent, as well as the specificity, of damage to simian virus 40 (SV40) DNA in lytically infected mammalian BSC-1 cells and in purified SV40 DNA preparations. C-1027 damage to intracellular SV40 DNA was quantitated by topological forms conversion analysis. A gradual decrease in intracellular supercoiled form I accompanied by an increase in form III was observed with C-1027 concentrations from 2 to 100 nM, with a 50% reduction in form I observed at 50 nM. Damage to purified SV40 DNA also was most pronounced between 10 and 100 nM C-1027. When concentrations were expressed as r values (drug/DNA molar ratio), the amount of C-1027 necessary to effect a 50% reduction in form I was lower for intracellular (r = 0.002) than for purified SV40 DNA (r = 0.0035). Double-strand damage was more likely to occur with C-1027 treatment of intracellular compared to purified SV40 DNA. However, with both purified and intracellular DNA, restriction enzyme digestion analysis revealed double-strand damage at a number of specific sites throughout the genome, particularly within the early region of the SV40 genome (e.g., within the coding sequence for large T-antigen). No significant damage was observed in either the origin (ORI) or the termination (TER) regions of SV40 replication. The extent of C-1027 damage to uninfected BSC-1 cell DNA was also quantitated using pulsed-field gel electrophoresis. At 0.1 nM (r = 2.8 x 10(-5), where incorporation of [3H]thymidine was reduced by 80%, 600 rad equiv of damage was detected in uninfected BSC-1 cells. At C-1027 r values from 1 x 10(-4) to 40 x 10(-4), double-strand breaks were from 80- to 40-fold more frequent in SV40 than in BSC-1 cell genomic DNA. By contrast, 50-fold more drug was necessary to inhibit intracellular SV40 DNA accumulation compared to [3H]thymidine incorporation into uninfected BSC-1 cells. Thus, SV40 DNA synthesis appeared to be less sensitive to C-1027-induced lesions than replication in uninfected BSC-1 cells.

Aminoglycosides↗

PubMed1981

The regulated expression of Epstein-Barr virus. III. Proteins specified by EBV during the lytic cycle.

The experiments show that 30 virus-induced or virus-specified proteins were synthesized in Raji cells after superinfection with Epstein-Barr virus (EBV) derived from P3HR1 cells. Using a combination of pulse labelling, application of cycloheximide blocks at different times post-infection, treatment with amino acid analogues and inhibition of DNA synthesis it was shown that three groups of proteins appear in Raji cells after superinfection; the synthesis of the proteins in any one group appears to be coordinately regulated. Amongst the six virus-induced proteins which were synthesized immediately after release from an early cycloheximide block one would expect to find those proteins essential for the transition from EBNA to EA synthesis. Using human sera with differing specificities for the various antigen groups 11 proteins were identified as being specifically precipitated by sera having high titres against the EBV-induced early antigen complex.

Antigens, Viral↗