- Volume 73, Issue 3, 1992
Volume 73, Issue 3, 1992
- Animal
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An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA
More LessThe product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as β-galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.
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β 2 Microglobulin on the envelope of urinary cytomegalovirus is not associated with host class I human leukocyte antigen α chain
More LessPrevious studies have shown that β 2 microglobulin (β 2m) is associated with glycoproteins present on the envelope of urinary human cytomegalovirus (CMV). β 2m is non-covalently associated with the α chain of human leukocyte antigen (HLA) class I antigens and therefore it was of interest to determine whether the class I α chain is also associated with the β 2m–CMV complex. Using a panel of monoclonal antibodies recognizing different conformational determinants on the HLA class I heterodimer or free β 2m, we have shown that β 2m but not the α chain of HLA class I could be immunoprecipitated from 125I-surface-labelled virions purified directly from urine. We therefore conclude that host class I HLA α chains are not associated with β 2m on the envelope of urinary CMV.
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Identification and control of the cis-acting elements of the immediate early gene of equid herpesvirus type 1
More LessConsensus cis-acting DNA sequences upstream of the immediate early (IE) gene of equid herpesvirus type 1 (EHV-1, strain Ab4) were identified. One copy of the conserved motif TAATGARATTC, which is the binding site for the host cellular factor Oct-1 and herpes simplex virus type 1 (HSV-1) virion protein, VmW65, complex, was identified at positions -630 to -620. Using transient transfections and chloramphenicol acetyltransferase assays the IE promoter of EHV-1 was shown to be trans-activated by VmW65 within the region -685 to +73. Ultraviolet light-inactivated EHV-1 was able to stimulate the expression of the IE gene of EHV-1 as well as HSV-1, indicating that EHV-1 possesses a protein equivalent to VmW65. The ubiquitous equid herpesvirus type 2 (EHV-2), which is not known to be a primary pathogen, was also able to trans-activate the EHV-1 and HSV-1 IE genes. Further work is being performed in order to identify the nature of the EHV-1 and EHV-2 trans-activating proteins.
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Characterization of the varicella-zoster virus gene 61 protein
More LessThe protein predicted to be encoded by varicella-zoster virus (VZV) gene 61 exhibits limited amino acid sequence similarity to the herpes simplex virus type 1 nuclear phosphoprotein Vmw110, which functions as a transcriptional activator. The gene 61 protein was expressed in its entirety, or as an amino- or carboxy-terminal fragment in Escherichia coli and vaccinia virus recombinants, and monospecific rabbit antisera were raised against an E. coli fusion between β-galactosidase and the majority of the gene 61 protein. Use of the antisera showed that the gene 61 protein is present in VZV-infected cell nuclei as a heterogeneous phosphoprotein of M r 62K to 65K. Phosphorylation occurs in the amino- and, to a lesser extent, carboxy-terminal portions of the protein. The carboxy-terminal region directs transport of the protein to the nucleus, whereas the amino-terminal region, which contains a potential zinc-binding domain, is responsible for a punctate distribution. Preliminary mapping data indicated that gene 61 is transcribed as a 1.8 kb mRNA which initiates about 65 bp upstream from the translation initiation codon, at a position located appropriately with respect to potential regulatory elements.
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On the cellular localization of the components of the herpes simplex virus type 1 helicase-primase complex and the viral origin-binding protein
More LessWe constructed recombinant viruses based on the herpes simplex virus type 1 mutant tsK which individually were able to express the products of four viral DNA replication genes (UL5, UL8, UL9 and UL52) in the absence of any of the other proteins required for viral DNA synthesis. These viruses were used in immunofluorescence experiments to investigate the cellular localization of the four replication proteins expressed. The results demonstrated that all three components of the viral helicase-primase complex (UL5, UL8 and UL52 proteins) must be co-expressed to allow their efficient localization to the nucleus. Since the UL5 and UL52 proteins together form a complex which is enzymatically indistinguishable from a complex formed from all three proteins, a possible role of the UL8 protein may be in facilitating nuclear uptake. The UL9 protein (origin-binding protein) efficiently entered the cell nucleus when expressed alone. Both UL9 protein and the tripartite helicase-primase complex exhibited patterns of fluorescence which resembled the ‘pre-replicative sites’ described previously.
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The myristylated virion proteins of herpes simplex virus type 1: investigation of their role in the virus life cycle
More LessHerpes simplex virus type 1 (HSV-1) gene UL11 encodes a myristylated virion protein. In this paper we have characterized the UL11 product further and investigated its role in the virus life cycle. Wild-type HSV-1 strain 17syn+ expresses three electrophoretically distinguishable UL11 polypeptide species. Analysis of single plaque isolates demonstrated that two virus populations exist within the 17syn+ stock: a major population encoding only the two higher M r species, and a minor population encoding the lowest M r species alone. DNA sequence analysis suggests that the latter polypeptide differs from the former ones at a single amino acid residue only. The UL11 polypeptides are synthesized as delayed early gene products and are phosphorylated in vitro. Following subcellular fractionation of infected cells, they are found predominantly associated with membranes. Within the virus particle, they appear to reside within the tegument. An insertion mutant containing the lacZ gene from Escherichia coli within the UL11 open reading frame is viable in tissue culture, although it gives smaller plaques and is impaired for growth compared to the wild-type parent or revertant viruses; it does not have a temperature-sensitive or host-range phenotype. Thus, although required for efficient replication, the myristylated HSV-1 virion protein, in contrast to those of many other viruses, is not essential for virus growth in tissue culture.
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A vaccinia serine protease inhibitor which prevents virus-induced cell fusion
More LessA deletion mutant lacking the non-essential vaccinia virus gene K2L, a member of the serine protease inhibitor superfamily, was constructed. This virus replicates in vitro in all cell types tested and its virulence and immunogenicity in vivo are comparable to those of the parent virus in intranasally inoculated mice. However, in a variety of cell lines the cytopathic effect of the deletion mutant (vKL4) is markedly different from that caused by the parent virus: the absence of K2L in infected cells results in extensive polykaryocytosis. Reinsertion of the K2L gene into vKL4 abolishes this fusion activity, thus confirming that the polykaryocytosis is the result of the deletion of K2L rather than of spontaneous mutations elsewhere in the genome, and that in cells infected with the WR strain of vaccinia virus the K2L gene product prevents fusion. The cell type-specific polykaryocytosis induced by vKL4 is apparent at late times post-infection, occurs from within and requires the synthesis of at least one late virus protein. Other vaccinia virus proteins known to be involved in fusion of infected cells are a 14K membrane protein which is required for fusion, and the haemagglutinin which prevents fusion. The haemadsorption properties of cells infected with the parent virus and the deletion mutant were indistinguishable: both haemadsorbed chicken erythrocytes. A monoclonal antibody against the 14K protein inhibited fusion of vKL4-infected cells, thus demonstrating that in addition to the absence of the K2L gene product, the 14K protein is required for fusion to occur.
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The predicted amino acid sequence of the spheroidin protein from Amsacta moorei entomopoxvirus: lack of homology between major occlusion body proteins of different poxviruses
More LessEntomopoxviruses replicate in the cytoplasm of insect cells and characteristically produce occlusion bodies which serve to protect the virion from the environment; the major component of these bodies is a protein called spheroidin. We have previously identified and sequenced the gene encoding the major occlusion body protein of eastern spruce budworm (Choristoneura biennis) entomopoxvirus (CbEPV) and found it to encode a 47K polypeptide which aggregates due to the formation of intermolecular disulphide bonds. In this publication we demonstrate that the insect poxvirus of Amsacta moorei produces spheroidin with a unit M r of 114.8K. The gene for this protein was cloned and sequenced, and the predicted polypeptide was demonstrated to contain 38 cysteine residues, a leucine zipper for possible protein-protein interactions and 14 potential Asn-linked glycosylation sites. Other than possessing a large number of sulphydryl groups, this protein showed no homology to its analogue found in cells infected with CbEPV. Antibodies directed against occlusion body proteins of the two viruses also failed to cross-react significantly on Western blots. In addition, nucleic acid probes prepared from the two different genes did not cross-hybridize on Southern blots of genomic DNA prepared from the viruses. Finally, the occlusion body proteins from the two insect viruses were compared with the A-type inclusion body protein of cowpox virus. Again, little homology between these proteins was evident, with the exception of a generally high cysteine content and a similarity between their late gene promoters. We conclude that the major occlusion body proteins of different poxviruses possess diverse primary structures, but all are capable of yielding large aggregates through the formation of disulphide bonds.
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Comparison of hantavirus isolates using a genus-reactive primer pair polymerase chain reaction
RNA of more than 40 hantavirus isolates, originating from rodents and humans of widely separated geographical areas, was copied to cDNA using reverse transcriptase and amplified by polymerase chain reaction (PCR). A genus-reactive oligonucleotide primer pair, flanking a 365 bp region of the G2 glycoprotein gene, was chosen for genus-reactive PCR. DNA products were digested with 20 restriction endonucleases and cleavage patterns were analysed. For strains of known sequence, the restriction patterns observed were consistent with those predicted from sequence data, demonstrating that the amplified products originated from target virus RNA. Further analyses suggested that all amplified viruses could be easily typed into one of five restriction patterns using only five enzymes. The categories identified by restriction analysis of PCR-amplified cDNA corresponded with serogroups established by plaque-reduction neutralization tests. This method may greatly simplify the identification of new hantavirus isolates.
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Degradation of cellular mRNA during influenza virus infection: its possible role in protein synthesis shutoff
More LessThe kinetics of cellular mRNA decay in influenza virus-infected cells have been studied by means of blot hybridization using as probes cloned cDNAs of α- and β-actin, α- and β-tubulin and vimentin. Both cellular mRNAs isolated from the cytoplasmic fractions as well as total cell mRNAs showed a rapid decay, with up to 50% concentration reductions at infection times at which influenza virus M1 mRNA was still not detectable. In contrast, these cellular mRNAs were stable in uninfected cells. To ascertain the possible role of mRNA degradation in the cellular protein synthesis shutoff, the kinetics of protein synthesis in infected cells were examined by two-dimensional gel electrophoresis of extracts pulse-labelled at several times after viral infection. The synthesis of the cellular proteins was reduced, showing kinetics paralleling those of mRNA decay. It is proposed that influenza virus infection induces the destabilization of mRNAs and that this mRNA degradation is, at least in part, responsible for cellular protein synthesis shutoff.
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Distribution and substrate specificity of intracellular proteolytic processing enzyme(s) for paramyxovirus fusion glycoproteins
Intracellular proteolytic processing of fusion glycoprotein precursors (F0) of paramyxoviruses, i.e. a virulent strain of Newcastle disease virus (NDV), parainfluenza virus type 3 (PIV3) and simian virus 5 (SV5), was examined in NALM6 and BSC40 cells and compared with that in LLCMK2 cells to investigate the distribution of the virus-activating protease(s) among the cells and its substrate specificity. BSC40 cells lack a processing endoprotease of the neuropeptide precursor, pro-opiomelanocortin (POMC), which possesses multiple cleavage sites at pairs of basic residues, Lys-Arg and Arg-Arg, a motif similar to that found in the cleavage site of the F0 proteins. In NALM6 cells, only small amounts of the F0 protein of virulent NDV was cleaved whereas those of PIV3 and SV5 were efficiently cleaved. In BSC40 cells the F0 proteins of these three viruses were cleaved normally as well as in LLCMK2 cells. The processing inhibitors monensin, chloroquine and A23187 suppressed the F0 cleavage in the three cell types. These results indicate that both NALM6 and BSC40 cells possess virus-activating proteases similar to that of LLCMK2 cells, but suggest that the enzyme of NALM6 may be slightly different in its substrate specificity from those of BSC40 and LLCMK2. The results also suggest that the virus-activating proteases are different in their distribution and substrate specificity from the processing enzyme of POMC.
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Location of antigenic sites defined by neutralizing monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide
More LessNeutralizing monoclonal antibodies directed against five antigenic sites on the spike (S) S1 glycopolypeptide of avian infectious bronchitis virus (IBV) were used to select neutralization-resistant variants of the virus. By comparing the nucleotide sequence of such variants with the sequence of the IBV parent strain, we located five antigenic sites on the amino acid sequence of the S1 glycopolypeptide. The variants had mutations within three regions corresponding to amino acid residues 24 to 61, 132 to 149 and 291 to 398 of the S1 glycopolypeptide. The location of three overlapping antigenic sites on the IBV spike protein was similar to the location of antigenic sites on the spike protein of other coronaviruses.
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Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients
More LessHeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz. Temperatures were kept below 30°C to prevent virus capsid modification. Under these conditions, the cell-associated virions remained fully infectious. Below 18°C, most of the viral label was recovered from a bottom region (BR) of the rate zonal gradients. Marker enzyme analysis and antibody accessibility showed that the BR consisted of virions bound to the plasma membrane. Between 18°C and 26°C, viral label also accumulated in a top region (TR) of the rate zonal gradients. According to the criterion of antibody accessibility, the virions associated with the TR were present within intracellular structures, probably lipid membranes. Electron microscopy confirmed the presence of vesicles and tubules in this region of the gradient. No correlation was found between the TR and endosomal, lysosomal or plasma membrane markers. The TR equilibrated at low density (1.10 g/ml) in Nycodenz (free virus, 1.31 g/ml). The results confirm that intact poliovirions can enter the cell and do so via lipid-bound vesicles.
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Poliovirus antigenic hybrids simultaneously expressing antigenic determinants from all three serotypes
More LessWe have constructed six hybrid polioviruses (PVs) modified to express PV type 2 and type 3 antigenic determinants on a PV type 1 (Mahoney) capsid. The hybrids were modified in neutralizing antigenic site (NAg) I and/or NAgII. They were viable, but impaired for growth in comparison to PV1 (Mahoney). Some hybrids modified to express type 2 and type 3 NAgI determinants simultaneously displayed some type 2 but no type 3 antigenicity (in addition to type 1 antigenicity associated with other antigenic sites). Hybrids modified to express a type 2 NAgI determinant and a type 3 NAgII determinant, or vice versa, displayed antigenic characteristics of all three serotypes, although expression of the modified NAgII determinant was weak. We conclude that it is possible to construct a viable hybrid PV simultaneously modified in NAgI and NAgII which expresses antigenic determinants of all three serotypes.
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Identification and characterization of foot-and-mouth disease virus O1 Burgwedel/1987 as an intertypic recombinant
More LessThe foot-and-mouth disease virus field isolate Burg-wedel/1987 subtype O1 was found to differ genetically from the antigenically related strain O1 Kaufbeuren within the region encoding the non-structural proteins. This genetic difference was indicated by the RNase mismatch cleavage method and confirmed by nucleotide sequencing. An alignment of sequences encoding proteinase 3C of the Burgwedel isolate and several other virus strains identified this isolate as an intertypic recombinant; the parent strains were O1 Kaufbeuren and a subtype C1 strain. Recombination occurred between nucleotide positions 5493 and 5521, within the region encoding peptide 3B1. Thus, the 5′ three-quarters of the O1 genome were fused to the 3′-terminal quarter of the C1 genome. Other contemporary isolates from the same district are not recombinants. Sequence alignment distinguished four patterns of proteinase 3C-coding sequences among the virus strains analysed: subtypes A12, C1 and O1 exhibit one pattern each, and another pattern is common to subtypes A5, A10 and O2.
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The nucleotide sequences of wild-type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant
More LessWe have shown previously that, compared to other enteroviruses, the coxsackievirus A9 (CAV-9) prototype strain, Griggs, contains a C-terminal extension to the capsid protein VP1 and that within this extension there is an RGD (arginine-glycine-aspartic acid) motif. To determine whether these features are found in other CAV-9 strains and therefore analyse whether they are likely to be functionally important, we have determined the nucleotide sequence of the appropriate region from five strains, isolated over a 25 year period. The results indicate that there is considerable diversity between the strains and there is little correlation between nucleotide sequence identity and date of isolation. All isolates exhibit the VP1 extension and although its amino acid sequence is otherwise variable, the RGD motif is common to all. This conservation of sequence, within a region which can otherwise vary, implies that the RGD sequence must be functionally significant. The VP1 extension shows similarity to sequences found in foot-and-mouth-disease virus strains and to part of the precursor of the cellular protein, human transforming growth factor β, and the possible significance of these observations is discussed.
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Shedding of a rhinovirus minor group binding protein: evidence for a Ca2+-dependent process
Soluble rhinovirus minor group binding activity was found to be shed into the medium upon incubation of HeLa cells at 37°C. Although substantial amounts of this protein were released, no decrease of virus binding to the cell surface was seen. When the membrane-associated receptor was stripped from the cells with trypsin, virus binding was rapidly restored from an intracellular pool even in the absence of de novo protein synthesis. The release of this 85K virus-binding activity was inhibited by metal chelators such as EDTA, EGTA or 1,10-phenanthroline. The potential involvement of a Ca2+-dependent protease and/or a phospholipase in this process is discussed.
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Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses
More LessNormal and rearranged RNA segments 10 of group A rotaviruses isolated from a chronically infected immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and were subsequently cloned and sequenced. Compared with the nucleotide sequence of the normal RNA segment 10, the rearranged form contains a partial non-coding duplication at its 3′ end and several point mutations. The normal RNA segment 10 was similar to that of bovine rotavirus.
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Autoprocessing of the human immunodeficiency virus type 1 protease precursor expressed in Escherichia coli from a synthetic gene
More LessA gene encoding an N-terminally extended precursor of 107 residues of the human immunodeficiency virus type 1 protease (PR107) was chemically synthesized and cloned into a bacterial expression vector, under the control of the araB promoter. PR107 was expressed alone or fused in phase to the amino or carboxy terminus of the bacterial β-galactosidase (β-gal). The yield of protease and β-gal was found to be significantly higher when the gene for PR107 was cloned upstream of the Escherichia coli lacZ gene (PR107–β-gal). Comparisons of the level of cloned protein expression between protease precursor and mature form suggested that this enhanced expression was due to the additional 5′ sequence of the PR107 gene, and occurred at the post-transcriptional level. Autoprocessing of protease precursor and its release from the β-gal fusion protein were analysed using wild-type and mutated cleavage sites. Mutations were introduced at amino acids downstream of the F–P scissile bond, at positions P4′ and P5′ in the C-terminal site (TLNF*PISP), and at position P3′ in a consensus N-terminal site (TLNF*PQITL) placed at the protease–β-gal junction. The data obtained suggested that (i) autoprocessing at the carboxy-terminal F–P bond was not significantly influenced by the presence of the N-terminal precursor sequence, (ii) P4′ and P5′ substitutions in the C-terminal site had no effect on cleavage, and (iii) P3′ in the N-terminal site tolerated a wide variety of substitutions.
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Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen
More LessWe have previously shown that immunization with solid matrix-antigen-antibody (SMAA) complexes induces both vigorous humoral and cell-mediated immune responses and have suggested that this method of vaccination may be developed for use in humans, and potentially as a vaccine against AIDS. Here we demonstrate that a small oligopeptide can act as a tag for the construction of SMAA complexes using a tag-specific monoclonal antibody and tag-linked antigens. We show that a 14-amino acid oligopeptide, present in the phospho (P) and V proteins of simian virus 5 (SV5), retains its antigenicity when attached to the C terminus of three ‘foreign’ proteins [p27 and gp110 of simian immunodeficiency virus (SIV) and glutathione S-transferase] such that these proteins can be incorporated into SMAA complexes using a monoclonal antibody (MAb) that was originally raised against the native SV5 P and V proteins. Mice were immunized with SMAA complexes containing recombinant p27-TAG and MAbs have been isolated that recognized native SIV p27. The significance of these results in terms of the development of SMAA complexes as human vaccines is discussed.
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Sulphate polyanions prolong the incubation period of scrapie-infected hamsters
The effect of the organic sulphated polyanions, pentosan sulphate (SP54), dextran sulphate 500 (DS500) and suramin, have been tested on golden Syrian hamsters infected with the 263K strain of scrapie by the intraperitoneal (i.p.) or the intracerebral route. SP54 had the greatest effect in prolonging the incubation period of the disease when administered within 2 h of the i.p. inoculum. The same amount of SP54 given 24 h after scrapie inoculation had a potent effect in some animals and no effect in others. This result suggests that SP54 inhibits the uptake of the scrapie agent into the nerve endings and/or carrier cells at the site of the inoculum, i.e. the peritoneum, and that this event occurs in about 24 h. DS500 had a similar although less potent effect (22.4 days delay during the incubation period) than SP54 (54.4 days) when administered within 2 h of scrapie injection by the i.p. route, and suramin had only a minimal effect (10 days). This study suggests that treatment of scrapie and related spongiform encephalopathies of animals and man is possible only before the agent has reached the clinical target areas of the brain.
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Demonstration of a hepatitis C virus-specific antigen predicted from the putative core gene in the circulation of infected hosts
An ELISA was used to detect a protein derived from the core gene of the hepatitis C virus (HCV) in human plasma. The solid phase antibody in the assay was a murine monoclonal antibody against a synthetic peptide deduced from the putative core gene of HCV (residues 39 to 74). An enzyme-labelled affinity-purified human antibody directed at another region within the HCV core (residues 5 to 23) was the second antibody tracer. The ELISA had a sensitivity capable of detecting a few ng/ml of the HCV core polypeptide expressed in Escherichia coli. Core antigen activity in plasma of infected hosts was detected after treatment of HCV RNA-rich fractions from buoyant density centrifugation with the detergent Tween 80. There was a direct correlation between core antigen ELISA values of a plasma fraction and intensities of polymerase chain reaction signals for HCV RNA. These observations are consistent with the proposal that the N-terminal sequence of the predicted polyprotein of HCV is a nucleocapsid protein, and that improved core antigen assays may correlate with viraemia.
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Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources
Based on variation in nucleotide sequence within restricted regions in the putative C (core) gene of hepatitis C virus (HCV), four groups of HCV have been postulated in a panel of 44 HCV isolates. They were provisionally designated types I, II, III and IV. A method for typing HCV was developed, depending on the amplification of a C gene sequence by polymerase chain reaction using a universal primer (sense) and a mixture of four type-specific primers (antisense). HCV types were determined by the size of the products specific to each of them. Type II was found in HCV samples from 131 (82%) of 159 blood donors, more often than in those from 48 (60%) of 80 patients with non-A, non-B (NANB) liver disease in Japan (P < 0.01). In 11 haemophiliacs who had received imported coagulation factor concentrates, type I was found in five, as against type II in four. Double infection with two different HCV types was found in two patients with chronic NANB liver disease (types I and II; II and III) and two haemophiliacs (types I and II; I and III). HCV types were identical in mother and baby in each of two examples of perinatal transmission, and were also identical in donor and recipient in a case of accidental needle exposure.
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Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction
More LessThe genomic RNA of hepatitis C virus (HCV) in the plasma of volunteer blood donors was detected by using the polymerase chain reaction in a fraction of density 1.08 g/ml from sucrose density gradient equilibrium centrifugation. When the fraction was treated with the detergent NP40 and recentrifuged in sucrose, the HCV RNA banded at 1.25 g/ml. Assuming that NP40 removed a lipid-rich surface coat from HCV, the 1.08 g/ml and 1.25 g/ml HCV RNA may correspond to intact HCV virions and nucleocapsids, respectively. The extraordinarily low density of the virion is unusual in comparison to the density of classified viruses.
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Baculovirus-expressed glycoprotein H of herpes simplex virus type 1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge
More LessWe have shown previously that herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) expressed by a baculovirus recombinant is transported to the cell surface in the absence of other HSV-1 gene products, and that the expressed gH has an apparent M r similar to that of authentic HSV-1 gH. We report here that antibodies raised in mice to this baculovirus-expressed gH neutralize the infectivity of HSV-1 in vitro; this neutralizing activity was not complement-dependent. Mice vaccinated with gH also developed delayed type hypersensitivity (DTH) to HSV-1. This is the first report of expressed HSV-1 gH inducing neutralizing antibody or DTH responses in vaccinated animals. In contrast to the gH expressed in mammalian systems, the ability of this baculovirus-expressed gH to induce a neutralizing antibody response may be due to the inability of the mammalian expression system to transport gH to the cell surface. Despite inducing anti-HSV-1 neutralizing antibody and DTH responses, vaccination of mice with gH did not protect the mice against lethal intraperitoneal challenge with HSV-1.
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The herpes simplex virus type 1 tegument protein VP22 is encoded by gene UL49
More LessVP22 is a major tegument protein of herpes simplex virus type 1 and is highly phosphorylated in the infected cell. Indirect evidence exists to suggest that it is encoded by gene UL49, present in the BamHI F fragment of the genome. Using the polymerase chain reaction we have cloned the UL49 open reading frame into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. After transfection into COS-7 cells expression of the gene product was detected by means of Western blotting and immunofluorescence. The results clearly indicate that the protein encoded by UL49 is VP22, and that in transfected cells it appears to have characteristics similar to those of the protein synthesized in infected cells.
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Comparison between in vitro neutralization titres and in vivo protection against homologous and heterologous challenge induced by vaccines prepared from two serologically distinct variants of foot-and-mouth disease virus, serotype A22
More LessGuinea-pigs were challenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaccination with baby hamster kidney (BHK) monolayer cell-adapted or BHK suspension cell-adapted strains of FMDV serotype A22 Iraq 24/64. The protection afforded by these vaccines was analysed as a function of antigen dose and the in vitro serum virus neutralization titres achieved. The results show that the level of neutralizing antibody induced that afforded 50% protection was similar for both vaccines in homologous or heterologous challenge situations. However, although the dose of antigen required to achieve this titre against homologous virus was similar for the two vaccines, approximately 20-fold more of the suspension cell-adapted virus was required to elicit a protective titre against heterologous challenge compared to the dose of monolayer cell-adapted virus required. A synthetic peptide representing the amino acid sequence 135 to 167 of VP1, which is identical in the A22 Iraq 24/64 variant viruses, was shown to induce protection against both homologous and heterologous virus challenge.
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Evolution of influenza B/Victoria/2/87-like viruses: occurrence of a genetically conserved virus under conditions of low epidemic activity
More LessNucleotide sequence analysis of the gene region coding for the HA1 domain of the influenza B virus haemagglutinin was performed on seven field strains isolated during the 1989 to 1990 season and two field strains isolated in 1985 and 1988 in Finland. All isolates were antigenically and genetically related to B/Victoria/2/87 virus and distinct from B/Yamagata/16/88 virus. The three strains isolated at the beginning of the 1989 1990 season in Turku were almost identical to an American variant (B/Texas/37/88-B/Ohio/10/88) of the previous season, whereas the four strains isolated later in the 1989 to 1990 season in Helsinki formed a new group of heterogeneous viruses. The phylogenetic tree compiled suggests that the two branches had evolved from a common origin, probably in 1987.
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Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus
More LessThe nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3′-terminal end of the M2 mRNA overlaps with the 5′ end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.
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Down-regulation of vesicular stomatitis virus transcription by the matrix protein of influenza virus
More LessThe matrix (M1) protein isolated from influenza A/WSN/33 virus, when reconstituted with ribonucleo-protein (RNP) cores of vesicular stomatitis virus (VSV), resulted in inhibition of VSV transcription in vitro. The presence of endogenous wild-type (wt) or mutant (tsO23) VSV matrix (M) protein on RNP cores did not prevent down-regulation of VSV transcription by reconstituted influenza virus M1 protein. In fact, endogenous VSV wt M protein augmented transcription inhibition by M1 protein reconstituted with RNP/M protein cores, whereas mutant tsO23 M protein endogenous to RNP cores had no effect on down-regulation of VSV transcription by M1 protein. These data suggest that VSV M protein and influenza virus M1 protein recognize two different sites on RNP cores responsible for down-regulation of VSV transcription. Monoclonal antibodies (MAbs) directed to epitope 2 of M1 protein had been previously shown to reverse transcription inhibition by M1 protein on influenza virus RNP cores, but the same epitope 2-specific MAb had little effect on transcription inhibition by M1 protein reconstituted with VSV RNP cores. VSV M protein bears a striking resemblance biologically and genetically to the M1 protein, including, as shown here, their capacity to bind viral RNA. However, the VSV wt M protein exhibited no capacity to down-regulate transcription by influenza virus RNP cores. The significance of these studies is the identification on VSV RNP templates of at least two separate sites for recognition of protein factors that repress VSV transcription.
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Viral RNA synthesis in tomato spotted wilt virus-infected Nicotiana rustica plants
More LessThe synthesis of viral RNA species in tomato spotted wilt virus-infected Nicotiana rustica plants was followed in terms of time and relative abundance. Systemic symptoms were visible after 4 days post-inoculation (p.i.), but viral (v) and viral-complementary (vc) strands of all three genomic RNA segments [large (L) RNA, medium (M) RNA and small (S) RNA] were detected from 2 days p.i. In addition, two subgenomic mRNAs, derived from S RNA, were detected. For the L RNA segment no subgenomic mRNAs were detected, suggesting that this segment is expressed via the synthesis of a genome-sized vc mRNA. A possible M-specific subgenomic mRNA was detected, showing a similar time course of appearance as the subgenomic mRNAs derived from the S RNA segment. Analysis of cytoplasmic RNA fractions revealed that both v and vc strands of all three genomic segments associate with the nucleocapsid protein into nucleocapsid structures, the vcRNA species being present in lower amounts. Intact, enveloped virus particles contained only the v strand of the L RNA segment and, surprisingly, both v and vc strands of the M and S RNA segment, though in different ratios.
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Epitope mapping on fragments of beet necrotic yellow vein virus coat protein
The location of five SDS-stable epitopes on the coat protein (CP) of beet necrotic yellow vein virus was determined by reacting Escherichia coli-expressed free CP, as well as fusion proteins (FP) containing fragments of the CP, with polyclonal and monoclonal antibodies on Western blots. Epitope 1, which has previously been found to be exposed on only one extremity of the virus particle, was located in the region between amino acids (aa) 1 and 7, i.e. on the N terminus of the CP. It was blocked when the N terminus of the CP was linked to a portion of the β-galactosidase sequence in an FP. Epitope 3, which has previously been found to be exposed on the opposite extremity of the particle, was located in the region between aa 37 and 59. Epitope 4, which is exposed along the entire length of the particle, occurs on the C terminus of CP (aa 183 to 188). Two previously unknown epitopes were identified in the regions between aa 115 and 125 and 125 and 140, respectively. The former was located on the same extremity of the particle as epitope 3, the latter became accessible only after denaturation of the particle. Nothing is known about the probably non-adjacent aa sequences that participate in the formation of the two SDS-labile epitopes (epitopes 2 and 5) which are found on one extremity and along the entire length of the particle, respectively.
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Nucleotide sequence analyses of peanut stunt cucumovirus RNAs 1 and 2
More LessThe nucleotide sequences of the RNAs 1 and 2 of the peanut stunt virus strain J (PSV-J) were determined and compared with those of the cucumber mosaic virus strain Y (CMV-Y, subgroup I), strain Q (CMV-Q, subgroup II) and the tomato aspermy virus strain V (TAV-V) at both the nucleotide and protein levels. RNA 1 of PSV-J consists of 3355 nucleotides (nt) and has one large open reading frame (ORF) which can encode the putative 1a protein of M r 112025. PSV-J RNA 1 and the la protein are 65 to 73% identical to those of CMV-Y and -Q, and 65 to 69% to those of TAV-V. RNA 2 of PSV-J contains 2946 nt and also has one large ORF which can encode the putative 2a protein of M r 93575. For RNA 2 and the 2a protein, identities between PSV-J and two strains of CMV are calculated to be 53 to 61%. When compared with TAV-V, the same degree of similarity as seen with CMVs is observed. The 1a protein has the consensus sequences found in some helicases and methyltransferases and the 2a protein includes a sequence which exists in several RNA-dependent RNA polymerases.
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Infectious in vivo transcripts of a plum pox potyvirus full-length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter
More LessA full-length cDNA clone of an aphid non-transmissible isolate of plum pox potyvirus (PPV) was rendered biologically active when placed under the control of the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase polyadenylation signal. The cDNA was constructed so that the exact 5′ end of the PPV RNA was present at the transcription initiation site. Inoculation of plasmid DNA onto Nicotiana benthamiana led to systemic infection, whereas local lesions were produced in Chenopodium amaranticolor and C. quinoa, typical of an infection with PPV. Examination of infected plants revealed PPV-specific virus particles as well as viral RNA, the coat protein and the non-structural large nuclear inclusion protein (NIb).
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A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA
More LessWe have synthesized and mapped a cDNA library representing the one major dsRNA element associated with hypovirulence in strain NB58 of the chestnut blight fungus, Cryphonectria (=Endothia) parasitica, which was isolated from recovering chestnut trees in New Jersey, U.S.A. The linear dsRNA has a size of approximately 12.5 kbp and is polyadenylated at the 3′ terminus of one strand. Molecular hybridization experiments indicate that there is sequence similarity between the NB58 dsRNA and dsRNAs from European isolates of C. parasitica, but not among dsRNAs of NB58 and those associated with other North American isolates. Hybridization experiments with mapped cDNA clones representing different regions of the 12.5 kbp dsRNA indicate that the termini and the 3′-proximal two-thirds (relative to the plus strand) are more conserved among NB58 and the European isolates than the rest of the 5′-proximal one-third. Nucleotide sequence analysis of the termini of NB58 dsRNA suggests common organizational features between it and the dsRNA from French-derived strain EP713.
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