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At least 865 records · Page 48Linked to original sources
PubMed2001

Dissecting the host response to a gamma-herpesvirus.

The murine gamma-herpesvirus 68 (MHV-68) provides a unique experimental model for dissecting immunity to large DNA viruses that persist in B lymphocytes. The analysis is greatly facilitated by the availability of genetically disrupted (-/-) mice that lack key host-response elements, and by the fact that MHV-68 is a lytic virus that can readily be manipulated for mutational analysis. The mutant virus strategy is being used, for example, to characterize the part played in vivo by an MHV-68-encoded chemokine-binding protein that may ultimately find an application in human therapeutics. Experiments with various -/- mice and monoclonal antibody depletion protocols have shown very clearly that type I interferons (IFNs) are essential for the early control of MHV-68 replication, while CD4+ T cells producing IFN-gamma function to limit the consequences of viral persistence. Virus-specific CD8+ effectors acting in the absence of the CD4+ subset seem initially to control the lytic phase in the lung following respiratory challenge, but are then unable to prevent the reactivation of replicative infection in epithelia and the eventual death of CD4+ T-cell-deficient mice. This could reflect the fact that the interaction between the CD8+ T cells and the virus-infected targets is partially compromised by the MHV-68 K3 protein, which inhibits antigen presentation by MHC class I glycoproteins. Immunization strategies focusing on the CD8+ T-cell response to epitopes expressed during the lytic phase of MHV-68 infection can limit virus replication, but are unable to prevent the establishment of latency. Other experiments with mutant viruses also suggest that there is a disconnection between lytic MHV-68 infection and latency. The massive nonspecific immunoglobulin response and the dramatic expansion of Vbeta4+ CD8+ T cells, which is apparently MHC independent, could represent some sort of 'smoke screen' used by MHV-68 to subvert immunity. Although MHV-68 is neither Epstein-Barr virus nor human herpesvirus-8, the results generated from this system suggest possibilities that may usefully be addressed with these human pathogens. Perhaps the main lesson learned to date is that all the components of immunity are likely to be important for the control of these complex viruses.

Animals↗
PubMed1987

Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication.

A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.

Animals↗
PubMed1993

Mutational analysis of the herpes simplex virus type 1 glycoprotein E promoter.

A set of linker-scanning mutations was constructed in the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promoter to define cis-acting regulatory elements common to late viral promoters. The gE promoter is a late viral promoter that has some activity in the absence of viral DNA replication and is representative of the gamma 1 class of HSV-1 promoters. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified two sequence elements, a TATA element and an element at the start of transcription, that corresponded to similarly positioned elements previously identified in the gC and gH late (gamma 2) HSV-1 promoters. Mutation of the initiation element reduced expression from this promoter during normal lytic infection, but had no appreciable effect on expression in the absence of viral DNA replication, suggesting a specific role in late gene expression. Analysis of expression from hybrid gE/gC promoters revealed that the TATA and the initiation elements of these two promoters were interchangeable and that expression from the gE promoter in the absence of viral DNA replication was due to regulatory elements upstream from TATA element.

Animals↗
PubMed1999

Characterization of natural Epstein-Barr virus infection and replication in smooth muscle cells from a leiomyosarcoma.

Cells from a leiomyosarcoma tumor (LMS-1) from a patient with the acquired immunodeficiency syndrome (AIDS) were explanted, cultured in vitro, and studied by phase-contrast microscopy for morphologic and growth characteristics, immunostaining for cell markers, EBER in situ hybridization and polymerase chain reaction for detection of Epstein-Barr virus (EBV), and immunostaining for expression of EBV antigens. The cells exhibited very slow growth in vitro, with unusual elliptical and spindle-shaped morphology and fragmentation of the cytoplasm into long, tapering, cytoplasmic processes. Greater than 90% of cells expressed diffuse distribution of the smooth muscle isoform of actin by immunoperoxidase staining. Approximately 25% of cells expressed very bright fluorescence by immunostaining of the smooth muscle isoforms of calponin and actin. The majority of cells demonstrated a weak signal for CD21; approximately 5-10% of cells showed a strong signal that was confined to cell surfaces. The cultured cells harbored EBV, and infectious EBV continued to be detected by polymerase chain reaction and virus culture through several passages in vitro. Several EBV antigens were expressed, including latent antigen EBNA-1, immediate-early antigen BZLF1, early antigen EA-D, and late antigens, including viral capsid antigen p160, gp125, and membrane antigen gp350. Human umbilical cord lymphocytes that were transformed with virus isolated from cultured cells yielded immortalized cell lines that expressed EBV antigens similar to other EBV-transformed lymphocyte cell lines. These results confirm that EBV is capable of lytic infection of smooth muscle cells with expression of a repertoire of latent and replicative viral products and production of infectious virus. EBV infection of smooth muscle cells may contribute to the oncogenesis of leiomyosarcomas.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Interferon cures cells lytically and persistently infected with African swine fever virus in vitro.

Human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) inhibit African swine fever (ASF) virus replication in Vero cells. IFN-alpha and IFN-gamma exert a synergistic inhibition. Human tumor necrosis factor (TNF) does not inhibit ASF virus replication in this cell line, but in combination with IFNs it has antiviral enhancing activity. Analysis of the mechanism of inhibition suggests that the action of these cytokines blocks a step that comes prior to DNA replication. The 2'-5' A synthetase activity is induced in Vero cells by treatment with these cytokines and is activated after ASF virus infection. More interesting is the finding that continuous treatment with IFN-alpha cures Vero cells from lytic and persistent infections with ASF virus. A potential application of IFN for the treatment of animals carrying the virus is suggested.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Large T antigen-rich viral DNA replication loci in SV40-infected monkey kidney cells.

The nuclear distribution of the large T antigen (T-Ag) during lytic infection of CV1 monkey kidney cells with SV40 virus was studied by immunoelectron microscopy. The viral protein was associated with the cellular chromatin and also accumulated within a small number of clearly delimited areas of the nucleoplasm. These T-Ag-rich areas were devoid of viral particles but contain 3-10 nm DNA filaments in an amorphous matrix. We have named these areas 'viral DNA/T-Ag loci.' The combination of the immunostaining for T-Ag with ultrastructural autoradiography revealed that these viral DNA/T-Ag loci were the sites of active SV40 DNA synthesis. We suggest that the viral DNA/T-Ag loci may represent definite structural domains specifically involved in viral DNA replication regulated by SV40-T antigen.

Animals↗
PubMed2004

Mechanisms of coxsackievirus B5 mediated beta-cell death depend on the multiplicity of infection.

Coxsackievirus infections may trigger and accelerate pancreatic beta-cell death, leading to type I diabetes. Unrestricted coxsackievirus B5 replication in cultured beta-cells inoculated with high multiplicity leads to rapid lytic cell death. Evidence from other virus-host cell systems indicates that host cell responses to infection may depend on the multiplicity of infection (MOI). Thus, the aim of this study was to compare the mechanisms of beta-cell death during high versus low multiplicity of coxsackievirus B5 infection. Cultures of highly differentiated mouse insulinoma cells and primary adult human islets were infected with coxsackievirus B5 at multiplicities of >1,000 or <0.5 TCID50 per cell. The results of nuclear morphology and viability stainings, TUNEL staining and electrophoretic DNA fragmentation analysis showed high multiplicity infection to predominantly induce necrosis and transient apoptosis. In low multiplicity culture, however, necrosis was only moderately induced and apoptosis increased steadily with time. This was best demonstrated by a tenfold higher apoptosis/necrosis ratio than after high multiplicity inoculation. Expression of gamma-glutamyl cysteine synthetase increased in both infective cultures but the level of intracellular glutathione permanently depleted only at high multiplicity and recovered fully at low multiplicity. Thus, apoptosis represents an important mechanism of beta-cell death after low multiplicity of coxsackievirus B5 infection. This process is associated with maintenance of a physiological intracellular glutathione profile differing dramatically from the high multiplicity infection during which necrosis dominates and intracellular thiol balance deteriorates. These data suggest that the pattern and mechanisms of coxsackievirus B5 infection induced beta-cell death depend on the MOI.

Animals↗
PubMedDate not supplied

The carrier state in foot and mouth disease--an immunological review.

The carrier state in foot and mouth disease (FMD) is characterized by the asymptomatic low-level excretion of foot and mouth disease virus (FMDV) from the oropharynx of ruminants for periods that are species and virus strain-dependent. Persistent infection with FMDV readily occurs following the failure of virus elimination at the acute stage of infection, a process thought to be mediated through the phagocytosis of antibody/virus immune complexes. Recent evidence supports the view that carrier cattle are important in the epidemiology of FMD in the field. The absence of histopathological change in persistently infected tissues and the reduced cytopathology of carrier virus isolates in tissue culture suggest that less lytic FMDV variants are generated or selected in the carrier animal. Altered virus replication, due to attenuation or interference, rather than antigenic variation may therefore allow evasion of the exaggerated FMDV-specific systemic and local humoral immune responses that occur in the carrier state. Although cell-mediated immune mechanisms for FMDV clearance have not been described, the eventual elimination of many persistent virus infections involves this arm of the immune system. Thus, further investigation of cellular elements of the immune response, more particularly the local interaction of mononuclear cell infiltrates with persistently infected cells, represents an area of research that has the potential to elucidate the carrier state problem.

Animals↗
PubMed2002

Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria.

The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

Bacteriophages↗
PubMed1984

Structural organization of human herpesvirus DNA molecules.

The herpesviruses are among the largest and most complex of all DNA viruses, and their genomes display an astonishing diversity in size, structure, and organization. In 1974, the features of large inverted repeats and structural isomerization were first discovered, and these proved to be characteristic properties of many herpesvirus genomes. Since then, research using the powerful techniques of modern molecular biology has revealed a great deal of comparative structural information about the arrangement of repetitive sequences and the location, structure, and primary nucleotide sequences of the genes for several easily assayed or abundantly expressed gene products. Extensive restriction enzyme cleavage maps and complete sets of cloned DNA fragments have been constructed for each of the five human herpesviruses, HSV-1, HSV-2, CMV, EBV, and VZV, and the entire 175,000-bp nucleotide sequence of EBV DNA has been determined. Based on these maps and reagents, the procedures of "DNA fingerprinting" and "dot hybridization" are proving useful at a clinical level for characterization of isolates and studying herpesvirus epidemiology. Strain differences, localized heterogeneity, tandem-repeat-defective genomes, and sites of cell-virus DNA homology have been described in some detail. The attention of basic researchers is now turning to equating structure with function, and rapid progress is expected in studies aimed at a better understanding of the mechanisms of viral DNA replication, maintenance of the latent state, reactivation, transformation, packaging, and regulation of the lytic cycle, etc using cloned functionally active DNA fragments, isolated intact genes and promoters, and DNA transfection and in vitro expression systems.

Base Sequence↗
PubMed2004

Brucella abortus rough mutants are cytopathic for macrophages in culture.

Rough mutants of Brucella spp. are attenuated for survival in animal models. However, conflicting in vitro evidence has been obtained concerning the intracellular survival of rough mutants. Transposon-derived rough mutants isolated in our laboratory were previously shown to exhibit small but significant reductions in intracellular survival in a 12-h in vitro assay. Several recent publications report that rough mutants exhibited increased macrophage uptake relative to their smooth parental strains, and a reduction in numbers at the end of the assay has been interpreted as intracellular killing. In an effort to explore the role of O antigen in the interaction between Brucella abortus and macrophages, we have monitored the uptake of rough mutants and survival in vitro by using the murine macrophage cell line J774.A1. The results confirm a 10- to 20-fold-increased uptake of rough mutants over that of smooth organisms under standard conditions. Recovery of the rough mutants persisted up to 8 h postinfection, but at the point when intracellular replication of the smooth organisms was observed, the number of rough organisms recovered declined. Fluorescence microscopy revealed the intracellular multiplication of both smooth and rough organisms, and assays performed in the absence of antibiotic confirmed the replication of the rough organisms. Examination by phase-contrast microscopy revealed the lytic death of macrophages infected with the rough mutants, which was confirmed by the release of lactate dehydrogenase (LDH) from the cell cytoplasm. Thus, the decline in the number of rough organisms was the result of the lysis of macrophages and not from intracellular killing. The cytopathic effect is characterized as necrotic rather than apoptotic cell death based on early LDH release, annexin V and propidium iodide staining, morphological changes of infected cells and nuclei, and glycine protection. The cytopathic effect was observed with macrophages at multiplicities of infection (MOIs) of as low as 20 and was not observed with epithelial cells at MOIs of as high as 2000. These findings suggest a role for O antigen during the early stages of host-agent interaction that is essential in establishing an intracellular niche that maintains and supports persistent intracellular infection resulting in disease.

Animals↗
PubMed1982

Precursor polypeptides to structural proteins of visna virus.

Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55(gag)). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55(gag) and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55(gag), is a likely precursor to the viral reverse transcriptase (Pr150(gag-pol)). Pr55(gag) and Pr150(gag-pol) are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150(env)). Pulse-chase experiments indicated that gPr150(env) matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150(env), which accumulated in the presence of 2-deoxy-d-glucose, appeared as a polypeptide of about 100,000 daltons. These results indicated that visna virus codes for the largest non-glycosylated env-related precursor among all of the retroviruses and therefore probably contains the largest env gene.

Animals↗
PubMed2001

Host range of small-ruminant lentivirus cytopathic variants determined with a selectable caprine arthritis- encephalitis virus pseudotype system.

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.

Amino Acid Sequence↗
PubMedDate not supplied

[Lambda plasmidophages and their properties].

Plasmidphage lambda NM::pBR322 has been constructed in vitro and characterized. Under normal conditions the hybrid DNA molecule undergoes a lytic cycle of phage development, whereas in the presence of antibiotic lambda DNA replicates in the cell extrachromosomally as a plasmid. Properties of plasmidphage lambda NM::pBR322 have been compared with the earlier constructed lambda gt::pMB9. It has been demonstrated that plasmid pMB9 in vivo can be precisely excised from the lambda gt::pMB9.

Bacteriophage lambda↗
PubMed2000

Molecular mechanisms of replication of herpes simplex virus 1.

The present review focuses on recent development in our understanding of the molecular mechanisms of DNA replication of herpes simplex virus 1 (HSV-1). Progress made in the characterization of the early events of viral DNA synthesis and of virus-host cell interactions, especially in the context with the formation of viral DNA replication sites, is highlighted. An up-dated overview is presented on important stages of lytic infection cycle, such as virion entry, viral DNA synthesis, viral DNA cleavage and packaging into preformed capsids; and maturation (nucleocapsid envelopment) and egress of virions. Many novel interactions are discussed that extend not only our knowledge of the biology of this virus but may represent possible new targets for antiherpesviral therapy.

Capsid↗
PubMed1990

A herpes simplex virus type 1 mutant containing a nontransinducing Vmw65 protein establishes latent infection in vivo in the absence of viral replication and reactivates efficiently from explanted trigeminal ganglia.

Vmw65, a herpes simplex virus type 1 (HSV-1) tegument protein, in association with cellular proteins, transactivates viral immediate early genes. In order to examine the role of Vmw65 during acute and latent infection in vivo, a mutant virus (in1814), containing a 12-base-pair insertion in the Vmw65 gene, which lacks the transactivating function of Vmw65 (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989) was examined in mice. Following corneal inoculation, the parental virus (17+) and the revertant (1814R) replicated effectively in eyes and trigeminal ganglia with 30 to 60% mortality. At either equal PFU or equal particle numbers, in1814 did not replicate in trigeminal ganglia and none of the infected mice died. Although in1814 did not replicate following corneal inoculation, it established latent infection in trigeminal ganglia. HSV-1 in1814 reactivated at explant as efficiently and rapidly as did 17+ and 1814R. Even low amounts of inoculated in1814 (10(2) PFU) were sufficient to establish latent infection in some animals. Since infectious in1814 was not detected at any time in mouse trigeminal ganglia, in1814 provided a unique opportunity to determine how soon after primary infection latency begins. Latent in1814 infection was detected shortly after virus reached the sensory ganglia, between 24 to 48 h postinfection. Thus, though Vmw65 may be required for lytic infection in vivo, it is dispensable for the establishment of and reactivation from latent infection. These data support the hypotheses that the latent and lytic pathways of HSV-1 are distinct and that latency is established soon after infection without a requirement for viral replication. However, the levels of Vmw65 reaching neuronal nuclei may be a critical determinant of whether HSV-1 forms a lytic or latent infection.

Animals↗