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At least 127 records · Page 7Linked to original sources
PubMed1996

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.

Animals↗
PubMed1969

Role of lysine in the replication of reovirus: I. Synthesis of complete and empty virions.

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm(3) and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm(3). The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm(3) was obtained from lysine-plus infected cells.

Journal Article↗
PubMed1980

Characterization of cricket paralysis virus-induced polypeptides in Drosophila cells.

Cricket paralysis virus purified from Galleria mellonella larvae was shown to be similar to virus purified from Drosophila melanogaster cells. Cricket paralysis virus contained three major structural polypeptides of similar molecular weight (around 30,000), had a buoyant density of 1.344 g/ml, and had a capsid diameter of 27 nm. Twenty virus-induced polypeptides could be detected in CrPV-infected Drosophila cells. Two major polypeptides found in the infected cells corresponded to two structural viral polypeptides (VP1 and VP3), whereas the third major intracellular polypeptide was the apparent precursor of the third viral structural polypeptide (VP2). Three of the primary virus-induced polypeptides had molecular weights of 144,000, 124,000, and 115,000. These and other polypeptides were chased into lower-molecular-weight proteins when excess cold methionine was added after a short [(35)S]methionine pulse. Although cricket paralysis virus has a number of characteristics in common with the mammalian enteroviruses, the extremely fast processing of high-molecular-weight polypeptides into viral proteins seems atypical. Also, no VP4 (8,000 to 10,000 molecular weight) has been found in the virus particles.

Journal Article↗
PubMed1988

Inoculation with BK virus may break immunological tolerance to histone and DNA antigens.

BK virus particles contain histones of host cell origin that combine with viral DNA to form minichromosomes. Data from earlier immunoblotting experiments demonstrated that rabbits inoculated i.v. with purified infectious BK virus produced antibodies not only to the viral structural protein VP1 but also to migrating polypeptides of a molecular mass ranging from 14-16 kDa. These proteins were believed to represent certain histone classes. To examine this hypothesis, sera from five rabbits inoculated with BK virus were analyzed by ELISA for antibodies against polynucleosomes, the individual histone classes, and double-stranded DNA, as well as against antigens carried by the structural viral proteins. Antibodies against polynucleosomes and also against histones H1 and H3 were found in sera from two of five inoculated rabbits. The same sera contained antibodies reacting with double-stranded DNA, whereas no antibodies against H2A, H2B, and H4 were detected. BK virus inoculation may thus lead to a break of tolerance, resulting in autoantibody production against highly conserved antigens that, in this context, may be regarded as "self-antigens", irrespective of the species in which they originate.

Animals↗
PubMed2005

Efficient axonal localization of alphaherpesvirus structural proteins in cultured sympathetic neurons requires viral glycoprotein E.

Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.

Animals↗
PubMedDate not supplied

[2 stages of influenza virus deproteinization in infected cells].

MDCK cells (a continuous cell line of dog kidney) were infected with 3H-uridine- or 14C-aminoacide-labeled influenza virus, the WSN strain. The cells were fractionated 30 min and 2 hours postinfection, and viral structures in subcellular structures were identified by sedimentation and density centrifugation and protein analysis in polyacrylamide gel. At 30 min after inoculation the perinuclear cytoplasm was shown to contain structures with the properties of viral nucleoids (ribonucleoproteins--RNP--surrounded with a layer of M protein). Their sedementation rate in glycerol gradients was 70--90 S, and the buoyant density in cesium chloride 1.30 g/ml. The nuclei were found to contain viral RNP with a buoyant density of 1.39--1.41 g/ml. Two hours after inoculation the amount of RNP in the nuclei decreased and in the cytoplasm increased indicating RNP transport from the nucleus into the cytoplasm. Treatment of the cells with cytochalasine B (a substance breaking nucleus-cytoplasm bonds) resulted in redistribution of radioactivity in subcellular fractions: an increase of radioactivity in the nuclear extract and a decrease in the nuclear sediment. Autoradiography of cytochalasine-treated cells revealed accumulations of viral structures around nucleoli. These data suggest participation of the nucleoli in transportation of viral structures into the cytoplasm. Based on the foregoing data, a hypothetical scheme of the processes of deproteinization and transport of viral structures in an infected cell has been proposed.

Animals↗
PubMed1971

Temperature-sensitive mutants of simian virus 40: infection of permissive cells.

Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants.

Animals↗
PubMed1993

Association of the non-structural P3 viral protein with cylindrical inclusions in potyvirus-infected cells.

Antibodies raised against the 42K non-structural P3 protein encoded by the RNA genome of a potyvirus (tobacco vein mottling virus, TVMV) have been used with immunogold labelling procedures to determine the subcellular location of P3 in infected Nicotiana tabacum cells. P3-specific gold label was found almost exclusively associated with the cylindrical inclusions typically formed in the cytoplasm of potyvirus-infected cells by another non-structural viral protein, the cylindrical inclusion protein. The P3 antibodies reacted with inclusions in both the transverse (pinwheel-like) and longitudinal (bundle-like) orientations of these structures. Immunocytological examination of TVMV-infected protoplasts showed the association of P3 with cylindrical inclusions during the early stages of formation of these structures and suggests that the P3 may be involved in the replication of potyviral RNA.

Genome, Viral↗
PubMed2001

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.

3' Untranslated Regions↗
PubMed1982

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.

Animals↗
PubMed1990

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus.

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.

Animals↗
PubMed1983

Molecular biology of rotaviruses. V. Terminal structure of viral RNA species.

The terminal structure of rotavirus genomic ds RNA and in vitro transcribed mRNA was investigated using S1 nuclease digestion and 3' terminal sequence analysis. S1 nuclease analysis indicated that viral mRNA was not a truncated copy of the genome RNA at its 5' end. The 3' terminal sequence analysis of genomic plus strands and mRNA showed that they were coterminal indicating that in vitro transcribed mRNA is a full length copy of the genomic template. The sequence analysis performed on isolated viral RNA species plus strands revealed a completely conserved 3' terminal octanucleotide 5'-AUGUGACC-3' present in both UK tissue culture adapted bovine rotavirus and a human rotavirus isolate. Sequencing of isolated genomic minus strands again revealed a completely conserved 3' terminal sequence of approximately the same length as that seen for the viral plus strands.

Base Sequence↗
PubMedDate not supplied

[Effect of remantadine on the deproteinization of influenza viruses in infected cells].

The effect of remantadine on penetration of influenza virus (the WSN strain) structures into MDCK cells was studied. Autoradiography of remantadine-treated cells infected with 3H-uridine-labeled virus showed remantadine to block the penetration of the parental viral structures from the perinuclear cytoplasm into the nuclei resulting in the structures aggregating in the perinuclear cytoplasm. Biophysical analysis of the viral structures recovered from the cells and analysis of their proteins by polyacrylamide gel electrophoresis showed that in remantadine-treated cells the amount of virus nucleoids (ribonucleoproteins, RNP, surrounded by a layer of M protein) increased in the perinuclear bytoplasm while the amount of intranuclear viral plasm while the amount of intranuclear viral RNPs decreased. These data suggest that the effect of remantadine is due to blocking of the second stage of deproteinization, that is, deproteinization of viral nucleoids to viral RNPs occuring in the perinuclear cytoplasm (possibly, on nuclear membrans); remantadine did not affect penetration of viral structures into the cells or the first stage of deproteinization (formation of nucleoids from virus particles). In order to inhibit deproteinization of viral structures remantadine should be present in the process of infection and be added soon after innoculation. When added 30 min after inoculation, remantadine has no effect on the intracellular distribution of viral structures.

Adamantane↗
PubMed1990

A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus.

A putative core protein derived from hepatitis C virus was expressed in E. coli. More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein. Western blotting with this E. coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.

Base Sequence↗
PubMed1989

Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus.

The nucleotide sequence of the gene that encodes for the structural viral protein VP1 of bovine rotavirus (RF strain) has been determined. The sequence data indicate that segment 1 contains 3302 bp and is A + T rich (65.3%). The positive strand of segment 1 contains a single open reading frame that extends 1088 codons and possesses 5'- and 3'-terminal untranslated regions of 18 and 20 bp, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 124,847, very close to the apparent molecular weight of VP1 (M.W. 125,000). The deduced amino acid sequence presents significant similarities with RNA-dependent RNA polymerase of several RNA viruses. VP1 was also synthesized in baculovirus using two transfer vecors: pAC461 and pVL941. Following infection of Sf9 cells with a recombinant baculovirus, a full-length nonfusion protein was synthesised which shares properties with authentic VP1 made in monkey kidney cells. The level of VP1 synthesis was about 10-fold higher when the baculovirus recombinant was derived from the pVL941 transfer vector. In that case, VP1 was expressed in yields approximately equivalent to 10% of the cellular protein. The recombinant protein was immunoprecipitated by hyperimmune serum raised against purified rotavirus. It also was immunogenic; a hyperimmune serum made in guinea pigs reacted with VP1 using immunoprecipitation and Western blot. This serum did not possess neutralization activity.

Amino Acid Sequence↗
PubMed2006

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

Antigens, Viral↗
PubMed1989

Comparisons of the structural proteins of avian infectious bronchitis virus as determined by western blot analysis.

The antigenic diversity of ten strains of avian infectious bronchitis virus (IBV) was examined by Western blot analyses using polyclonal antisera specific for the Massachusetts 41 (M41), Gray, Arkansas DPI (Ark DPI), Connecticut (Conn) and Australian T (Aust T) serotypes. Although antigenic variation was found in all three structural viral proteins, the matrix protein appeared to be antigenically the most highly variable. Four distinct antigenic groups, which did not correspond to virulence or pathotype, could be defined according to the variations observed in the matrix protein. Somewhat less variation was seen in the spike polypeptide. The only variation in the nucleocapsid protein was indicated by the lack of a detectable reaction between the Aust T antiserum and the Ark DPI nucleocapsid protein. Antisera made against M41 had the broadest reactivity while antisera against Aust T, the only strain tested which was exotic to the U.S.A., had the greatest specificity.

Animals↗