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PubMed1979

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.

Alpharetrovirus↗

PubMed1976

Analysis of structural polypeptides of purified human cytomegalovirus.

Human cytomegalovirus strain C87 was purified by the following procedures. (i) Extracellular virus was concentrated by centrifugation at 100,000 X g for 90 min and passed through a Bio-Rad Bio-Gel A-15m column. Most of the virus was recovered in the void volume. (ii) After two consecutive isopycnic potassium tartrate gradient centrifugations (20 to 50%), coinciding peaks of plaque titer, protein, and radioactivity were found at a density of from 1.20 to 1.21 g/cm3. To characterize the structural polypeptides of human cytomegalovirus and to establish relative purification criteria, virus was purified from two mixtures: (i) [35S]methionine-labeled extracellular virus mixed with an equal volume of unlabeled normal culture fluid; (ii) unlabeled extracellular virus mixed with an equal volume of [357a1methionine-labeled normal culture fluid. The extent of purification, as judged by the ratio of cellular to viral radioactivity, was 39-fold; i.e. about 2.5% of the protein in the purified virus preparation could be accounted for by host protein contamination. Electrophoresis of purified [35S]methionine-labeled virus on a polyacrylamide gel slab showed that there were at least 33 viral structural polypeptides (VPs), and their molecular weights ranged from 11,000 to 290,000. Autoradiograms obtained from electropherograms of purified [14C]glucosamine labeled virus showed six bands. Four of these were so broad that several VPs corresponded to each of the glycosylated bands. When heavy (two fractions close to 1.21 g/cm3) and light (two fractions close to 1.20 g/cm3) fractions of the PFU peak from the second potassium tartrate gradient were analyzed separately, the number of polypeptides observed was the same, but the relative amounts of some polypeptides differed. The major polypeptide, VP17, was found in greater amounts in the heavy fraction (35%) than in the light fraction (22%). The amount of DNA as a percentage of the weight of protein was 2% for the light fraction and 1% for the heavy fraction.

Cytomegalovirus↗

PubMed1996

Respiratory syncytial virus G-protein modulates cytokine release from human peripheral blood mononuclear cells.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children and adults. In vivo the host has to cope with intact replicative virus, with non-replicative virus, and/or with viral structural proteins including the outer membrane G-protein. We analyzed the role of purified RSV G-protein with regard to its modulatory efficacy for interleukin (IL) -10, IL-12, and tumor necrosis factor-alpha (TNF-alpha) release from human peripheral blood mononuclear cells (PBMC). These cytokines seem to contribute to the deleterious effect in viral infections. Treatment of PBMC with RSV at a multiplicity of infection of 10 down to 0.001 induced the release of TNF-alpha, IL-10, and IL-12; also time kinetics and dose-responses differed markedly. Stimulation of PBMC with purified RSV G-protein (from 0.001 up to 10 microgram/10(6) PBMC) led only to a pronounced increase in IL-10 within a concentration range from 0.01 up to 0.5 microgram/10(6) PBMC with a maximum between 12 and 18 h of incubation. AT later time points (24, 48, and 72 h) G-protein concentrations above 1 microgram/10(6) PBMC suppressed IL-10, TNF-alpha, and IL-12 release from human PBMC. Coating of PBMC with RSV G-protein suppressed IL-10, TNF-alpha, and IL-12 release after subsequent stimulation with RSV. Our data indicate a regulatory role of RSV G-protein immune responses toward viral infection.

Cells, Cultured↗

PubMed1995

Occurrence and role of an early antigen and evidence for transforming ability of porcine circovirus.

By means of indirect immunofluorescence assay (IFA) using natural swine immune serum and hyperimmune serum from rabbits infected with porcine circovirus (PCV), a PCV antigen was detected present prior to the onset of viral and cellular DNA synthesis in nucleoli of cells of synchronized and growth stimulated infected PS cell cultures grown for more than 12 h in the presence of hydroxyurea. The number of cells containing specifically fluorescing nucleoli increased with increasing time of growth in the presence of hydroxyurea. The concomitant increase in the number of cells containing virus structural (VS) antigen in the nuclei and the increase in the amount of replicative (RF) DNA and accompanying 5 S DNA after release from the hydroxyurea block suggest that EA is involved in induction of PCV DNA replication. Primary pig kidney cell cultures persistently infected with PCV survived mock-infected control cultures for 16 passages. They had lost contact inhibition and formed cell colonies in soft agar at a ratio of 0.1 to 0.4%. Cell lines derived from agar colonies showed properties of transformed cells e.g. low requirement for serum growth factors, ability to overgrow a continuous cell layer, anchorage independence of growth. In transformed cells stimulated to growth and grown in the presence of hydroxyurea, non-structural viral antigen visible by IFA in nucleoli and VS antigen located in the cytoplasm were expressed. Contrary to virus bound nuclear VS antigen in productive infection, accumulation of cytoplasmatic VS antigen was independent of DNA synthesis and caused cell destruction, thus limiting growth of cell layers and colonies in soft agar.

Animals↗

PubMedDate not supplied

Biological characteristics of rhabdoviruses in different host cells.

Rhabdoviruses are a group of viruses capable of infecting vertebrates, invertebrates and plants and showing a wide range of host-parasite relationships. Biological characteristics of viruses can vary according to the type of cells in which they grow. The Authors analyze differences in viral structure and multiplication related to the host. Some final considerations on the evolution of this group of viruses are also reported.

Cells, Cultured↗

PubMed1982

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells.

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.

Animals↗

PubMed2006

Hsp70 negatively controls rotavirus protein bioavailability in caco-2 cells infected by the rotavirus RF strain.

Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.

Caco-2 Cells↗

PubMed1976

Isolation and characterization of a virus associated with progressive pneumonia (maedi) of sheep.

A virus with growth and morphologic characteristics of progressive pneumonia (maedi-visna) virus was isolated from the lungs of sheep with typical clinical and postmortem changes of chronic progressive pneumonia. The virus grew slowly in cultures of embryonic ovine lung cells, causing syncytial formation and degeneration. Syncytia developed much slower and involved fewer cells than reported for other similar viruses isolated from sheep. As seen with the electron microscope, the virus reproduced by budding from cell surfaces. Two types of virions were seen-a large particle (120 to 140 nm) with an electron-lucent center and dense laminated outer rim, and a small particle (80 to 110 nm) with an electron-dense core surrounded by a single membrane. Viral structures and fragments similar to the large extracellular particles were seen in the cytoplasm of a few cells. These characteristics are reported for other viruses isolated from sheep with progressive pneumonia.

Animals↗

PubMed2005

Quantification of rotavirus-like particles by gel permeation chromatography.

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.

Animals↗

PubMed1983

Visualization of antigens attached to cytoskeletal framework in animal cells: colocalization of simian virus 40 Vp1 polypeptide and actin in TC7 cells.

Actin and the simian virus 40 viral structural polypeptide Vp1 are observed to be present on cytoskeletal fibers of virus-infected TC7 cells, when these antigens in detergent-extracted whole cell mounts were labeled by specific antibodies and colloidal gold particles coated with a second antibody. In both cases, actin and Vp1 were found associated with fibers and fiber-associated electron-dense materials. Patches or clusters of colloidal gold particles denoting the presence of either Vp1 or actin were found on fibers uniformly distributed throughout the cytoplasm. By using simultaneous decoration of the two antigens with colloidal gold particles of different diameters, it was shown that the majority of Vp1 appears attached to cytoskeletal fibers in association with cellular actin. When Vp1 and actin were decorated with Imposil and ferritin simultaneously in infected cells that were fixed first and then permeabilized with saponin, both labels were found in the same spatial domain of the cell cytoplasm. Thus, the colocalization of Vp1 and actin on the cytoskeletal framework seems to reflect their actual state in the living cells. The electron-dense material to which colloidal gold particles localize in our cytoskeletal preparations may be the remnants of subcellular structures with which actin and Vp1 are both associated in intact cells.

Actins↗

PubMed1987

Inhibitory effect of papaverine on HVJ (Sendai virus) replication in rat glioma C6 cells.

The replication of HVJ (Sendai virus) in C6 rat glial cells was found to be inhibited by treatment of the cells with papaverine, an inhibitor of cAMP phosphodiesterase, but not with cAMP or dibutyryl cAMP. In addition, cyclic GMP which often manifests a reciprocal relationship to cAMP did not counteract the inhibition of HVJ yield by papaverine. Both viral genome replication and transcription were suppressed slightly by treatment of the cells with papaverine. In the cells cultured in the presence of papaverine, the synthesis of viral proteins and their phosphorylation occurred at normal rates. Membrane immunofluorescence and cell surface immunoprecipitation showed that the viral glycoproteins HN and F0 were expressed on the cell surface of the papaverine-treated cells. Moreover, all the viral structural proteins were associated with plasma membrane isolated from the treated cells. These results indicate that papaverine treatment suppresses some part of the process of virus budding at the plasma membrane.

Animals↗

PubMed2003

Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

Antiviral Agents↗

PubMed1983

Circulating antibodies to the feline leukemia virus and FOCMA in cats.

Presence of antibodies to the structural proteins of the feline leukemia virus and the feline oncornavirus-associated cell membrane antigen FOCMA was analyzed in sera of 120 cats from household environments. In our experiments 35% of the sera tested were positive with the viable cells of FL-74 feline lymphoma cell line. On the other hand, only 5% of the feline sera reacted with FeLV from FL-74 cells in the solid phase RIA. Selected positive sera were analyzed for the presence of antibodies to the viral structural proteins of FeLV and FOCMA in lysates of FL-74 cells by radioimmunoprecipitation. Absorption of the selected sera (reacting with viable FL-74 cells) with disrupted FeLV had a negligible effect on the precipitating activity of the 70000 protein.

Animals↗

PubMed2001

Equine rhinitis A virus: structural proteins and immune response.

Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.

3C Viral Proteases↗

PubMed1996

Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase.

The Nef protein of primate immunodeficiency viruses is essential for establishing a highly productive pathogenic infection in vivo. In tissue culture, Nef is not required for infection but enhances viral infectivity. This effect is most pronounced in unstimulated primary lymphocytes and occurs in the early phase of infection prior to viral gene expression. Since Nef expression does not lead to obvious changes in virus composition, it was of interest to analyze whether Nef is incorporated into virus particles. Here, we show that Nef is specifically immunoprecipitated from radioactively labeled human immunodeficiency virus type 1 (HIV-1)-infected cells and virus particle preparations. Quantitative analysis revealed Nef to be incorporated on the order of 10% of reverse transcriptase incorporation, which corresponds to 5 to 10 molecules of Nef per virion. In infected cells, Nef was detected as a full-length 27-kDa protein. In contrast, approximately 50% of particle-associated Nef corresponded to an 18-kDa species which comigrated with the larger product after in vitro cleavage of purified HIV-1 Nef by the viral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Nef is cleaved concomitantly with viral structural proteins on maturation of virus particles. This cleavage is likely to be functionally significant because it dissociates the conserved core domain from the N-terminal membrane attachment region. Our results suggest that the profound influence of Nef on establishing infection of unstimulated cells in tissue culture and in vivo is mediated by virion-associated Nef which functions in early infection before viral gene expression.

Animals↗

PubMed1972

Development of coliphage T5: ultrastructural and biochemical studies.

Electron microscopic studies of Escherichia coli infected with bacteriophage T5(+) have revealed that host nuclear material disappeared before 9 min after infection. This disappearance seemed to correspond to the breakdown of host deoxyribonucleic acid (DNA) into acid-soluble fragments. Little or no host DNA thymidine was reincorporated into phage DNA, except in the presence of 5-fluorodeoxyuridine (FUdR). Progeny virus particles were observed in the cytoplasm 20 min postinfection. Most of these particles were in the form of hexagonal-shaped heads or capsids, which were filled with electron-dense material (presumably T5 DNA). A small percentage (3 to 4%) of the phage heads appeared empty. On rare occasions, crystalline arrays of empty heads were observed. Nalidixic acid, hydroxyurea, and FUdR substantially inhibited replication of T5 DNA. However, these agents did not prevent virus-induced degradation of E. coli DNA. Most of the phage-specified structures seen in T5(+)-infected cells treated with FUdR or with nalidixic were in the form of empty capsids. Infected cells treated with hydroxyurea did not contain empty capsids. When E. coli F was infected with the DO mutant T5 amH18a (restrictive conditions), there was a small amount of DNA synthesis. Such cells contained only empty capsids, but their numbers were few in comparison to those in cells infected under permissive conditions or infected with T5(+). The cells also failed to lyse. These results confirm other reports which suggest that DNA replication is not required for the synthesis of late proteins. The data also indicate that DNA replication influences the quantity of viral structures being produced.

Bacteriolysis↗

PubMed1990

Identification and stability of a 30-kDa nonstructural protein encoded by mRNA 2 of mouse hepatitis virus in infected cells.

A bacterial expression vector encoding a fusion protein containing almost the entire first open reading frame (ORF1) of mRNA 2 of MHV-A59 has been constructed. The purified fusion protein was used to raise antibodies to the protein encoded by mRNA 2 ORF1. Specificity of the antibodies was verified by immunoprecipitation of the in vitro translation product of ORF1, which was reconstructed downstream of a T7 promoter. In vivo the antiserum reacted specifically with a 30-kDa protein synthesized in MHV-A59- and MHV-JHM-infected cells. This 30-kDa protein could not be identified in purified virions and is therefore a nonstructural viral protein. The expression pattern of this 30-kDa nonstructural viral protein in infected cells was shown to be identical to that of the viral structural proteins. However, in comparison to the nucleocapsid protein pulse-chase studies revealed a relative short half life for this 30-kDa protein in vivo.

Animals↗

PubMed1975

Simian hemorrhagic fever virus: a new togavirus.

Using the prototype strain of SHF virus, we have confirmed the nature of the genome (RNA), the presence of an envelope (derived from an internal membrane), the virion size (45-50 nm), and probable cubic symmetry. We have also described four viral structural proteins and determined the phospholipid content of the virions. The known properties of SHF virus suggest that it should be classified in the Togavirus family, and possibly in the Flavivirus group.

Animals↗