- Volume 76, Issue 12, 1995
Volume 76, Issue 12, 1995
- Animal
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Characterization of the baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain
More LessThe DNA sequence and transcription pattern of the p10 gene from the spruce budworm baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV) were analysed. The CfMNPV p10 gene codes for a protein 81 amino acids in length: this is the shortest p10 peptide identified thus far. A novel characteristic of the CfMNPV p10 gene is that it contains tandem late initiation sites (TAAG) having different temporal transcription patterns. Comparative analysis of CfMNPV p10 and the amino acid sequences of other p10 gene products showed that they each appear to have a similar N-terminal structure: an amphipathic alpha-helical terminus which condenses as coiled-coil multimers. Another feature of the p10 N terminus is that the central region of the coiled-coil domain resembles a bend or hairpin loop and could fold into a hairpin or form a bent rod structure. The condensation of p10 monomers to coiled-coil multimers is likely to be a step leading to the formation of p10 fibrous bodies in infected cells.
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Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus
More LessThe Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted M r of 22000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M r of 24000, in close agreement with the molecular mass predicted from the nucleotide sequence. G22 is not significantly homologous to any known protein, nor is a G22 homologue present in the Autographa californica MNPV (AcMNPV). Temporal expression studies indicated that the G22 gene was transcribed at readily detectable levels in the presence of cycloheximide. Transcripts were detected immediately after the virus adsorption period and throughout the infection cycle. The early transcriptional start sites of G22 map to a sequence that resembles a subset of RNA polymerase II promoters/start sites that are found upstream of Drosophila melanogaster developmental and retrotransposon genes which lack TATA box motifs. Several consensus late baculovirus promoter/mRNA start site sequences (ATAAG) were identified upstream of the G22 gene start codon.
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Expression and localization of a baculovirus protein phosphatase
More LessWe have characterized the expression of a baculoviral gene, ptp, and determined the location of its gene product, a protein tyrosine/serine phosphatase (BV-PTP), during virus infection. Using an antibody raised to a BV-PTP fusion to glutathione S-transferase, we found that ptp was expressed as a 19 kDa polypeptide at late times during virus infection. However, we also found that BV-PTP was present in the virions of both the budded and occluded forms so that a low level of BV-PTP is also present at the beginning of the infection process. Biochemical fractionation also showed that BV-PTP was primarily localized to the cytoplasm in transfected cells but that BV-PTP was present in both the cytoplasm and the nucleus of baculovirus-infected cells. Immunoelectron microscopy revealed that BV-PTP was associated with fibrillar structures which form in both the cytoplasm and the nucleus of baculovirus-infected cells.
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The ATP-binding and ATPase activities of human papillomavirus type 16 E1 are significantly weakened by the absence of prolines in its ATP-binding domain
More LessThe E1 protein of human papillomavirus (HPV) type 16 is the only known papillomavirus E1 which does not contain any proline residues in the phosphate-loop (P-loop) of its ATP-binding site. To ascertain whether this feature influences the activities of HPV-16 E1, we generated a mutant HPV-16 E1 (E1Pro) in which prolines are inserted in place of alanines in this site, making the P-loop identical to its bovine papillomavirus type 1 counterpart. Glutathione S-transferase (GST) fusion proteins (GSTE1wt and GSTE1Pro) were produced, purified and used to assay for ATP-binding ability, ATPase activity and ability to complex with the HPV-16 E2 protein. The results show that the lack of prolines in the P-loop, which is unique to HPV-16 E1, significantly weakens its ATP-binding and ATPase activities without affecting its ability to complex with the HPV-16 E2 protein.
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A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers, plasmids and bacterial hosts
More LessVaccinia virus is one of the most widely applied expression systems for use in eukaryotes in molecular biology. Expression of heterologous genes in the vaccinia virus system, however, requires integration of the foreign DNA into the vaccinia virus genome by means of homologous recombination or by direct molecular cloning. In both cases, plasmid vector constructs are required that contain the gene of interest and, usually, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified vaccinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any further cloning steps. The gene of interest is amplified by PCR using oligonucleotide primers that provide an SfiI site at the 5′ end and an RsrII site at the 3′ end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the unique SfiI/RsrII sites of the modified vaccinia virus genome. Using this approach, intermediate plasmid constructs and bacterial hosts are not required and time consuming screening steps can be omitted, because 90% of the virus progeny carry the foreign DNA.
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Release of extracellular particles by recombinant vaccinia virus over-expressing the major envelope protein p37K
More LessDuring the replication cycle of vaccinia virus, four different forms of viral particles are produced. The two extracellular enveloped forms, cell-associated enveloped virus and extracellular enveloped virus, are responsible for cell-to-cell transmission and long-range spread of infection both in vivo and in vitro. Despite the biological importance of the enveloped forms, the mechanism of envelopment and the components involved in this process have been analysed only recently. Therefore the individual steps and the rate-limiting factors of the envelopment process are still unknown. The protein p37K, an unglycosylated but acylated envelope protein of molecular mass 37 kDa, has been shown to be essential for envelopment. However, this study shows that over-expression of p37K by vaccinia virus recombinants reduces rather than increases the yield of infectious enveloped virus which is mainly due to the enveloped virions exhibiting a strongly diminished specific infectivity.
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Genomic analysis of a transposition-deletion variant of orf virus reveals a 3.3 kbp region of non-essential DNA
More LessRestriction endonuclease analysis of the DNA extracted orf virus strain NZ2, which had been serially passaged in primary bovine testis cells, revealed a population of variants that had over-grown the wild-type virus. At least three distinct mutant forms were identified in which the right end of the genome had been duplicated and translocated to the left end, accompanied by deletions of sequences at the left end. Sequencing of a single variant isolated from the heterogeneous population revealed that recombination had occurred between non-homologous sequences. In this case, 6.6 kb of DNA at the left end of the genome had been replaced by 19.3 kb from the right end. The transposition resulted in the deletion at the left end of 3.3 kb of DNA encoding three genes and the terminal sequence of a fourth gene. The three genes completely deleted were a homologue of dUTPase, a gene that encodes a protein containing ankyrin-like repeats and a homologue of the 5K gene of the vaccinia virus WR strain. Experimental inoculation of sheep showed that the genes are also non-essential in vivo, but that the size of the lesion was reduced, compared with that induced by the wild-type, and resolved more rapidly.
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Immunomodulation of peripheral T cells in chickens infected with Marek’s disease virus: involvement in immunosuppression
More LessMarek’s disease virus (MDV) causes T cell immunosuppression in chickens during latent infection. Morphological changes specific to apoptosis were demonstrated in peripheral blood mononuclear cells (PBMC) of MDV-infected chickens at 2–3 weeks post-inoculation (p.i.). Analysis of DNA fragmentation in T cell subsets in the peripheral blood revealed that CD4+ T cells but not CD8+ T cells underwent apoptosis after MDV infection. The proportion of CD4+ T cells, but not that of CD8+ T cells, in the peripheral blood expanded transiently at 16 days p.i., and rapidly decreased 1 week later. The decrease in CD4+ T cells might be mediated by apoptosis, because a rapid reduction in CD4+ T cells was observed when these cells underwent apoptosis. Analysis of the T cell-receptor (TCR) repertoire of the peripheral blood showed that Vβ1 but not Vβ2-αβ TCR-bearing cells expanded at 16 days p.i., when the transient expansion of the CD4+ T cell population was observed in these chickens. Flow cytometric profiles also showed that the expression of CD8 was down-regulated after infection with MDV, but there was no difference in the expression level of CD4 molecules between normal and infected chickens. Northern blot analysis indicated that the down-regulation of CD8 occurred at the transcriptional level. These results suggest that both apoptosis of CD4+ T cells and down-regulation of CD8 molecules could contribute to the immunosuppression caused by MDV.
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Cytokines restore MHC class I complex formation and control antigen presentation in human cytomegalovirus-infected cells
More LessCD8+ cytotoxic T cell (CTL) clones with specificity for defined minor and major histocompatibility (H) antigens were used to monitor antigen presentation in human cytomegalovirus (HCMV)-infected skin fibroblasts. At the immediate early stage of virus replication antigen presentation was intact, but was abolished during the early and late phase. Lack of CTL recognition was not selective for certain antigens but was associated with decreased steady state levels of nascent MHC class I complexes and unassembled MHC class I heavy chains, whereas free β2-microglobulin remained abundant. HCMV also affected the stability of both immature endoglycosidase H (Endo H)-sensitive and mature Endo H-resistant MHC class I molecules, suggesting that the virus interferes with antigen presentation at more than one step during maturation of the MHC class I complex. The action of interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) lifted the block of MHC class I complex formation by stimulating synthesis, assembly and stability of MHC class I molecules. This resulted in restored antigen presentation provided that cells were exposed to the factors before HCMV infection. Because few MHC molecules suffice for CTL recognition these cytokines compensated for the negative viral effect on the antigen presentation function. Nevertheless, the viral interference with MHC class I complex formation was still active. The data imply that specific cytokines limit the immune evasion potential of HCMV from CD8+ T lymphocyte control.
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Mutations within conserved motifs in the 3′–5′ exonuclease domain of herpes simplex virus DNA polymerase
More LessWe investigated mutations within the presumed 3′–5′ exonuclease domain of the DNA polymerase from herpes simplex virus type 1. The mutation sites correspond to residues in DNA polymerase I (Escherichia coli) which bind two metal ions that are required for exonuclease function. To evaluate the effect of the herpesvirus mutations on enzymatic activity, we over-expressed the wild-type DNA polymerase and one mutant enzyme using a baculovirus expression system. Both proteins exhibited DNA polymerase activity after partial purification, but the mutant protein was drastically deficient in exonuclease activity. This finding suggests that the herpesvirus exonuclease may utilize the same metal-ion-mediated mechanism employed by DNA polymerase I. We also attempted to transfer each of the mutations into the herpesvirus genome using a marker rescue protocol. Although wild-type sequences could be transferred readily, recombinant viruses carrying mutant sequences were not recovered. We discuss the possibility that the mutations are lethal and suggest mechanisms by which a deficiency in 3′–5′ exonuclease might cause loss of viability.
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Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays
More LessRecombinant baculoviruses have been constructed which express the hepatitis C virus (HCV) NS3 proteinase and its substrates in insect cells. The expressed proteinase has been shown to carry out trans-cleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in a cell-based assay. When assayed in a cell-free system using in vitro translated substrates, the proteinase could perform Trans-processing of the NS4A/4B and NS5A/5B junctions, but only when coexpressed with NS4A, either as an NS3-4A precursor or by co-infection of cells with NS3- and NS4A-expressing recombinant baculoviruses. Possible reasons for the absolute requirement of the NS3 proteinase for NS4A in vitro are discussed.
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In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses
By the use of recombinant baculoviruses, the trans-cleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein. Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also found that the partially purified NS3 serine proteinase propared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained will provide basic knowledge on processing of the HCV polyprotein.
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Expression of hepatitis C virus envelope proteins in transgenic mice
In an attempt to establish a model for hepatitis C virus (HCV) infection, we produced mice transgenic for the HCV envelope genes, E1 and E2, under the control of a regulatory element from hepatitis B virus. F1-generation mice from the established founders demonstrated expression of both E1 and E2 proteins as glycosylated forms in their organs including the liver. Immunostaining revealed the localization of envelope proteins principally in the cytoplasm of hepatocytes around the hepatic central veins. Furthermore, E1 and E2 proteins were shown by immunoprecipitation to be associated with each other in the mouse liver. There was no evidence of tissue pathology in the mouse liver up to 16 months of age, suggesting that E1 and E2 proteins per se may not have direct pathogenic effects. Our results demonstrate the first successful expression of HCV gene products in the mouse liver and the association of in vivo expressed HCV envelope proteins. This transgenic mouse would be a good model to study the virus-cell interactions of HCV such as intracellular transport and assembly of envelope proteins. Also it may be a good model system to determine the role of these proteins in the pathogenesis of hepatitis or extra-hepatic manifestations in HCV infection by the introduction of cytotoxic T lymphocytes specific for the envelope proteins.
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Classical swine fever virus-specific cytotoxic T lymphocytes and identification of a T cell epitope
More LessClassical swine fever virus (CSFV) -specific cytotoxic T lymphocytes (CTL) were derived from peripheral blood mononuclear leukocytes of immunized NIH-minipigs (MHC d/d haplotype) after in vitro restimulation with infectious CSFV. Their cytotoxic activity was determined against CSFV-infected target cells obtained from simian virus 40 (SV40) large T antigen-transfected immortalized kidney cells of a syngeneic miniature swine. Experiments with separated effector cell populations revealed that the CSFV-specific cytotoxic activity was mediated by CD4−CD6+CD8+ MHC class I-restricted T lymphocytes. Infection of target cells with various vaccinia virus/CSFV recombinants led to the identification of a major antigenic site for CSFV-specific CTL near the cleavage site between the non-structural proteins p80 (NS3) and p10 (NS4a). Using synthetic overlapping nonapeptides which covered this protein region the sequence ENALLVALF is the first sequence to be identified as an MHC class I-restricted T cell epitope recognized by CSFV-specific CTL.
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The phosphoprotein gene of a dolphin morbillivirus isolate exhibits genomic variation at the editing site
More LessThe nucleotide sequence of the phosphoprotein (P) gene of a dolphin morbillivirus (DMV) isolate was determined. Like those of other morbilliviruses the DMV P gene encoded P and C proteins in overlapping open reading frames and V protein by editing the P gene transcript. Among P mRNA based clones the editing site variants GGGC, GGGG, GAGC and GGGGGGC predicting a P protein, and the variants GGGGC and GGGGG predicting a V protein, were found. Surprisingly, the three variants GGGC, GGGG and GAGC were also found among clones generated from genomic RNA of the DMV isolate. Thus, more than one viral genome type appeared to be present in cells infected with the DMV isolate. By a similar analysis of the virus genomes in the tissue from which the DMV isolate was obtained, only the GGGC type was found, indicating that the GGGG and GAGC types arose during adaptation of the virus to growth in cell cultures. No editing site variants likely to have arisen by editing the GAGC type were encountered, and it remains to be determined whether mRNA encoding V protein can be transcribed from genomes with this editing site. Using antisera raised against the common N terminus and unique C termini of the predicted P and V proteins, the in vivo expression of these proteins was demonstrated.
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A region of the coronavirus infectious bronchitis virus 1a polyprotein encoding the 3C-like protease domain is subject to rapid turnover when expressed in rabbit reticulocyte lysate
More LessIn order to investigate the mechanisms involved in the processing of infectious bronchitis virus polyproteins, several candidate regions of the genome have been cloned and expressed in vitro. During these studies it was observed that the translation product encoded by one of these clones (pKT205) was poorly expressed. Biochemical and genetic analyses revealed that the basis for the poor expression was a post-translational event involving ubiquitination of the protein and degradation by an ATP-dependent system operating in the reticulocyte lysate used for the in vitro expression. Two independently acting regions which conferred instability were identified, one of which mapped to the predicted 3C protease domain, contained within the 5′ end of the clone, while the other, more C-terminal region, was effective in conferring instability upon a heterologous protein to which it had been transferred. These regions may influence the stability of the authentic viral protein(s) in vivo and hence allow for the control of their expression and/or function at the level of proteolysis by cellular protease(s).
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Editing efficiency of hepatitis delta virus RNA is related to the course of infection in woodchucks
More LessBased on evidence in vitro which shows that the small form of hepatitis delta virus (HDV) antigen (S-HDAg) initiates virus replication, whereas the long form (L-HDAg) encoded by the edited L-genome inhibits replication, we first put forward the hypothesis that HDV RNA editing efficiency, i.e. the intracellular L/S-genome ratio, could be a determining factor on the course of the infection. In order to analyse the precise sequence of events after infection, woodchuck carriers of woodchuck hepatitis virus (WHV) were superinfected with HDV and sequential changes in HDV RNA editing efficiency were analysed in relation to the duration of viraemia. Our findings show that: (1) in both transiently and persistently viraemic woodchucks, the percentage of L-genome is higher at the early stage of onset of the disease than at the late stage; (2) at the early stage of onset the percentage of L-genome is higher in cases with transient viraemia than in those with persistent viraemia; (3) a relatively greater decrease in L-genome is seen later in transiently viraemic animals than in those that remain persistently viraemic. In view of the above findings in vivo and other supporting evidence in vitro, we propose a hypothesis for the pathogenesis of HDV. This hypothesis predicts the outcome of acute infection and we suggest a gene therapeutic approach to this disease based on the intracellular accumulation (or increase) of the L-genome.
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Genetic divergence of poliovirus strains isolated in the Karachi region of Pakistan
More LessSeventy-seven wild poliovirus strains isolated from poliomyelitis cases in the Civil Hospital of Karachi in Pakistan in 1989–1993 were selected for partial sequence analysis covering the VP1/2A junction region of the viral genome to study the genetic relationships and epidemiological links between strains. Viral RNA was partially amplified by RT-PCR and sequenced by a solid phase method. Computer analysis revealed genetic divergence of the strains within each serotype. Most of the nucleotide differences between strains were silent: only a few specific amino acid substitutions were seen in the sequenced region. Three genotypes of poliovirus type 1 and two of poliovirus type 3 were co-circulating, while type 2 strains were represented by a single genotype. Representatives of all the genotypes present have been found among previously or concurrently characterized strains isolated elsewhere, but direct epidemiological links were found only in the case of serotype 1. Many of the epidemics caused by poliovirus type 1 in other countries were genetically linked to Pakistan. This study clearly shows the endemic circulation and wide variation of all three poliovirus serotypes in southern Pakistan and indicates the need for more effective vaccination programmes to prevent the further spread of these wild viruses.
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Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system
More LesscDNA cassettes encoding the foot-and-mouth disease virus (FMDV) structural protein precursor (P1-2A) together with the 3C protease, which cleaves this molecule to 1AB, 1C and 1D, were constructed. These cassettes were introduced into vaccinia virus (VV) transfer vectors. Attempts to isolate recombinant VVs constitutively expressing these cassettes were unsuccessful. However, when the P1-2A-3C cassette was placed under the control of the bacteriophage T7 promoter, stable VV/FMDV recombinants were isolated. Co-infection with recombinant VV vTF7-3 (which expresses T7 RNA polymerase) led to the production of correctly processed FMDV capsid proteins. Analysis by sucrose gradient centrifugation showed that material which co-sedimented with natural empty capsid particles (70S) was formed. Electron microscopy revealed empty capsid-like particles with diameters of about 30 nm. Studies using monoclonal antibodies specific for conformational epitopes indicated that the antigenicity of the synthetic particles was similar to whole virions and natural empty capsid particles. Surprisingly, merely the modification of a single amino acid residue within the myristoylation consensus sequence at the N terminus of P1-2A allowed the isolation of a recombinant VV which constitutively expressed the correctly processed proteins. However, the capsid proteins expressed from this mutant cassette failed to assemble into 70S empty particles.
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Identification of the location of antigenic sites of swine vesicular disease virus with neutralization-resistant mutants
More LessNeutralization sites on swine vesicular disease virus (SVDV) have been identified by sequence analysis of neutralization-resistant mutants. Eight neutralizing monoclonal antibodies (MAbs) were produced and neutralization-resistant mutants were selected with the MAbs. Resistance of the mutants to neutralization was shown using the stab-neutralization method, and the results indicated the presence of five neutralization sites on the virus. The location of each site was identified from amino acid changes resulting from nucleotide substitutions in the mutants, and designated site 1 (residues 87 and 88 of VP1), 2a (residue 163 of VP2), 2b (residue 154 of VP2), 3a (residues 272 and 275 of VP1, 60 of VP3) and 3b (residues 70 and 233 of VP2, 73 and 76 of VP3). The locations of the amino acid substitutions at each site formed a cluster on a computer-simulated three-dimensional model of SVDV and were exposed to the surface of the virion.
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Development and characterization of a novel xenograft model permissive for human papillomavirus DNA amplification and late gene expression
Human papillomaviruses (HPVs) are important human pathogens associated with a range of epithelial neoplasia. The rising incidence of HPV infection and association of HPV with malignancy has led to increased interest in appropriate management of these infections. Development of new therapies for viral warts has been frustrated by the lack of availability of models permissive for viral replication. Here we describe the development of a HPV-severe combined immunodeficient mouse model which reproduces mature HPV-infected epithelia. Grafting of anogenital and laryngeal papillomas harbouring either HPV-6 or HPV-11 resulted in the formation of a differentiated neo-epithelium exhibiting the hallmark features of HPV infection including basal hyperplasia, acanthosis and koilocytosis. The reformed warty epithelium contained amplified HPV DNA and expressed capsid protein in the differentiated layers. A striking feature is the production of macroscopic papillomata in an anatomically relevant and accessible site, providing a system of particular relevance for the temporal evaluation of developing lesions and selection of antiviral agents.
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The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons
More LessNeural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.
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Thermosensitive UL9 gene function is required for early stages of herpes simplex virus type 1 DNA synthesis
More LessDNA replication of herpes simplex virus type 1 (HSV-1) is dependent on a virus-encoded sequence-specific origin-binding protein, the product of the UL9 reading frame. We have identified the mutations in the UL9 gene of three temperature-sensitive (ts) mutants of HSV-1 which are responsible for the ts phenotype (A90T in mutant tsS and V220M in tsR and tsX). The mutations are located in two different conserved helicase sequence motifs of UL9. Two further alterations (I204T and E280D) compared to the published sequence were found in the mutant, revertant and parental wild-type strain 17syn + sequences and therefore seemed to be irrelevant for the ts phenotype. The ts function of the UL9 protein was required at early times during DNA synthesis whereas upward temperature shifts at later times did not considerably inhibit DNA synthesis.
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The origin-binding domain of the herpes simplex virus type 1 UL9 protein is not required for DNA helicase activity
More LessThe UL9 protein of herpes simplex virus type 1 binds to specific sequences within the viral origins of DNA replication and also functions as a DNA helicase. The C-terminal 317 amino acids of the 851 residue protein specify sequence-specific binding to the viral origins and the N-terminal 400 contain several motifs characteristic of many DNA and RNA helicases. To investigate whether the origin-binding domain is required for helicase function we have expressed a truncated version comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells infected with the recombinant virus and chromatographed over ATP-agarose. DNA helicase, DNA-dependent ATPase and a novel single-stranded DNA-binding activity were present in fractions containing the truncated UL9 protein but not in corresponding gradient fractions from a control virus infection. These results indicate that the DNA helicase function of UL9 does not require the presence of the origin-binding domain and suggest that an interaction between the N-terminal domain and distorted or partially single-stranded regions of DNA may play a role in unwinding the origin region.
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The cell-coded polypeptide U90 increased by herpes simplex virus type 2 infection induces Fos and DNA synthesis
More LessLytic infection with herpes simplex virus type 2 (HSV-2) induces synthesis of a cell-coded protein of molecular mass 90 kDa, termed U90. U90, whose expression is specific in tumour cells is, in addition, excreted from the cell. To determine if the function of U90 could be advantageous for the virus the protein was purified from the medium of HSV-2 infected cells, shown to be similar to the internalized form and its effects on cell growth studied. Addition of U90 to quiescent fibroblast cells stimulated the expression of Fos, the product of the cellular transcription factor c-fos and increased the incorporation of [3H]thymidine into DNA, factors which may facilitate HSV-2 replication. However, U90 might also induce abnormal cells such as those in precancerous lesions to proliferate.
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Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice
The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of value as a component of an HSV multi-subunit vaccine.
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Co-expression of the Epstein—Barr virus BXLF2 and BKRF2 genes with a recombinant baculovirus produces gp85 on the cell surface with antigenic similarity to the native protein
More LessGlycoprotein H (gH) is a conserved herpesvirus gene product functionally implicated in the penetration of the virus into the host cell. Other human herpesviruses require an accessory glycoprotein named gL for the synthesis of mature gH. We constructed a series of recombinant baculoviruses to determine whether gL expression can overcome the constraints upon Epstein—Barr virus (EBV) gH (gp85) folding and transport in this expression system. When gH and gL were co-expressed some EBV gH was transported to the insect cell surface. Deletion of 27 amino acids from the gH carboxy terminus resulted in the secretion of an 80 kDa protein (gHt) into the culture medium when it was expressed either in the presence or absence of gL and this protein could also be immunoprecipitated with E1D1. In contrast, gL was not secreted into the culture medium. Our results suggest that either co-expression of gH with gL or removal of the predicted transmembrane anchor sequence can overcome some of the constraints upon EBV gH expression in the baculovirus system.
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Replication-defective adenovirus type 5 as an in vitro and in vivo gene transfer vector in chickens
More LessThe capacity of E1A gene-deleted and thus replication-defective adenovirus type 5 (Ad5) to transduce foreign genes in chicken embryo fibroblasts (CEF) as well as in chickens was investigated. The lacZ and luciferase genes were successfully transduced in CEF by replication-defective Ad5, demonstrating that these cells possess receptor(s) for binding and penetration of Ad5. A single intramuscular inoculation of Ad-gD, a replication-defective Ad5 harbouring the gD gene of pseudorabies virus, in adult and 1-day-old chickens led to the production of very high titres of specific antibodies. These gD-specific antibodies persisted for at least 56 days. These results demonstrate that replication-defective Ad5, despite its mammalian origin and the deletion of the E1A gene, is a good candidate for developing non-spreading vaccines in poultry.
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The precore/core promoter mutant (T1762A1764) of hepatitis B virus: clinical significance and an easy method for detection
More LessRecently, a new hepatitis B virus (HBV) mutant with HBe antigen-negative phenotype has been characterized, in which one TATA box-like motif of the precore/core promoter had degenerated: most frequently by both A → T and G → A mutations at positions 1762 and 1764, respectively. The clinical significance of this mutant is as yet unknown. In our present study, the T1762 A1764 mutant was sought in sera from HBV-infected blood donors and chronic liver disease patients by directly sequencing a PCR-amplified region of HBV DNA. Also, because the A1764 mutation generates a Sau3AI cleavage site (GGTC → GATC), we digested the PCR products with Sau3AI to see if cleavage would occur at this specific site. Our results mostly corroborated the earlier report but we found a higher-than-predicted frequency of HBe antigen-positive blood donors positive for the mutant (22%). The titres of HBe antigen in these mutant-positive sera were slightly decreased compared to the titres in wild-type HBV infection. In addition, these blood donors had relatively high (though within the normal range) serum alanine aminotransferase (ALT) levels, suggesting that the T1762 A1764 mutation could be used as a sensitive laboratory marker for insidious hepatitis in these otherwise ‘asymptomatic’ carriers. The Sau3AI assay, which is much more convenient than sequencing, was shown to be useful for the detection of the T1762 A1764 mutant in an extensive number of clinical samples.
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Targeted infection of a retrovirus bearing a CD4-Env chimera into human cells expressing human immunodeficiency virus type 1
More LessWe constructed a hybrid Moloney murine leukaemia virus (MoMLV) bearing both a chimeric CD4 and the wild-type MoMLV envelope protein (Env) on its surface. The chimeric molecule, consisting of a surface domain of CD4 and the C-terminal two-thirds of MLV Env, was expressed on the cell surface. When expressed in MoMLV-infected cells, the CD4-Env chimera was incorporated into the virion as efficiently as the wild-type MoMLV Env. The hybrid MoMLV could infect human HeLa cells (although not with high efficiency) only if the cells were expressing human immunodeficiency virus type 1 genome. This method of ligand incorporation into a virion may lead to a development of a cell-specific retroviral vector for targeting gene therapy.
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Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation
X.-F. Yu, Z. Matsuda, Q.-C. Yu, T.-H. Lee and M. EssexLentiviral Gag polyproteins have a proline-rich protein, p6, at their C terminus. There are conflicting reports about the function of p6 in virus release. In the present work, mutants that affect p6 of human immuno-deficiency virus type 1 (HIV-1) Gag polyprotein were constructed and analysed. None of the mutants prevented virus release completely; however, detachment of budding particles was less efficient as evidenced by electron microscopy. Virions of the p6 truncation mutant B2TAA had a significantly reduced number of Pol proteins (p66, p51 and p34) and an increased amount of incompletely processed Gag proteins compared with the parental virus. A mutation that altered the cleavage site between p6 and p1 did not significantly affect virus assembly, virus release or protein processing with the exception of cleavage between p6 and p1. However, virions of this mutant (B2P6C) exhibited irregularshaped core structures that were distinct from the cone-shaped core structure seen in the parental virion. B2P6C mutant virus was non-infectious in CD4+ T cells. These results suggest that mutations in p6 affect efficient detachment of budding particles from the cell surface. Proper cleavage between p6 and p1 may be critical for the formation of the distinctive cone-shaped core structure of HIV-1 virions.
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Sequence comparison of open reading frames 2 to 5 of low and high virulence United States isolates of porcine reproductive and respiratory syndrome virus
More LessThe sequences of ORFs 2 to 5 of five United States (US) porcine reproductive and respiratory syndrome virus (PRRSV) isolates with differing virulence were determined. The nucleotide and deduced amino acid sequences of these isolates were compared with those of other known PRRSV isolates. The amino acid sequence identity between seven US PRRSV isolates was 91–99% in ORF 2, 86–98% in ORF 3, 92–99% in ORF 4 and 88–97% in ORF 5. The low virulence US isolate had highest sequence variation in ORFs 2 to 4 compared to the other US isolates. A hypervariable region with antigenic potential was identified within the major envelope glycoprotein. Phylogenetic analysis of ORFs 2 to 7 indicated the existence of at least three minor genotypes within the major US genotype. The low virulence US isolate formed a branch distinct from the other US isolates. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.
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A minimal and optimal cytotoxic T cell epitope within hepatitis C virus nucleoprotein
Amino acid residues 81–100 of the hepatitis C virus (HCV) nucleoprotein contain a cytotoxic T cell epitope that is recognized by cytotoxic T lymphocytes (CTLs) in association with human leukocyte antigen B44. With panels of truncated and overlapping peptides, the minimal and optimal epitope recognized by CTLs was shown to be a 9-mer peptide (residues 88–96). The peptide can stimulate effectively CTLs that are able to recognize endogenously synthesized and processed HCV nucleoprotein.
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Isla Vista virus: a genetically novel hantavirus of the California vole Microtus californicus
Prospect Hill virus (PH) was isolated from a meadow vole (Microtus pennsylvanicus) in 1982, and much of its genome has been sequenced. Hantaviruses of other New World microtine rodents have not been genetically characterized. We show that another Microtus species (the California vole M. californicus) from the United States is host to a genetically distinct PH-like hantavirus, Isla Vista virus (ILV). The nucleocapsid protein of ILV differs from that of PH by 11.1% and a portion of the G2 glycoprotein differs from that of PH by 19.6%. ILV antibodies were identified in five of 33 specimens of M. californicus collected in 1975 and 1994–1995. Enzymatic amplification studies showed that 1975 and 1994–1995 ILV genomes were highly similar. Secondary infection of Peromyscus californicus was identified in Santa Barbara County, California. A long-standing enzootic of a genetically distinct hantavirus lineage is present in California voles.
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Detection of measles virus nucleoprotein mRNA in autopsied brain tissues
More LessBy means of RT–PCR, a portion of measles virus (MV) mRNA encoding nucleoprotein (NP) could be detected in 11 (18%) of 61 brain tissue samples obtained from administrative autopsy cases, who apparently had not suffered from subacute sclerosing panencephalitis (SSPE)-like central nervous system disorders. Most of the brain-derived NP sequences showed significant asynonymous nucleotide substitutions when compared with wild-type MV isolates and SSPE virus. Our present results suggest that MV commonly persists in the human brain without causing apparent clinical symptoms, probably due to decreased virus replication.
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Sequence analyses of human parainfluenza virus type 4A and type 4B fusion proteins
cDNAs encoding human parainfluenza virus type 4A and type 4B (hPIV-4A and -4B) fusion (F) proteins were cloned and sequenced. The predicted amino acid sequences of the F proteins had similar characteristic traits to those reported for the F proteins of other paramyxoviruses. They were more closely related to the F proteins of simian virus 5 (SV5), mumps virus (MuV), hPIV-2 and Newcastle disease virus (NDV) than to the F proteins of hPIV-1, hPIV-3, Sendai virus (SV) and measles virus (MV). In addition, hPIV-4A, hPIV-4B, SV5 and MuV shared a common feature of genomic organization: there was a small ORF between the F and haemagglutinin—neuraminidase (HN)-coding sequences, implying a common ancestry.
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The relative amount of an influenza A virus segment present in the viral particle is not affected by a reduction in replication of that segment
More LessThe principles of influenza A virus replication and packaging are not fully understood. In order to investigate the signals required for these processes we have introduced mutations in the terminal non-coding region of an influenza A virus neuraminidase (NA) gene. Specifically, we have obtained two viruses, NA/X and NA/Y, which produce a reduced amount of NA-specific genomic RNA in infected cells but not in the viral particle. These data indicate that (i) specific signals which affect the amount of RNA in the viral particle are distinct from those required for viral replication and (ii) the amount of packaged RNA is not strictly dependent on the amount of RNA produced during replication. In addition, mutant NA/Y was shown to be effectively attenuated in mice. Thus, diminished replication of one viral segment might be a principle on which to base a live influenza virus vaccine.
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Genome organization of ageratum yellow vein virus, a monopartite whitefly-transmitted geminivirus isolated from a common weed
More LessA full-length copy of a single genomic component of the whitefly-transmitted geminivirus ageratum yellow vein virus (AYVV) has been cloned from an extract of infected Ageratum conyzoides originating from Singapore. Sequence analysis shows that the genomic component encodes two virion-sense (V1 and V2) and four complementary-sense open reading frames (C1-C4), typical of DNA A of whitefly-transmitted geminiviruses from the Eastern hemisphere. A genomic component equivalent to DNA B was not detected in extracts of infected A. conyzoides. The cloned genomic component produced a systemic infection in Nicotiana benthamiana, Phaseolus vulgaris and Lycopersicon esculentum when introduced into plants by agroinoculation, and symptoms were identical to those produced by wild-type virus introduced into these hosts using viruliferous whiteflies. However, attempts to re-establish a systemic infection in A. conyzoides either by agroinoculation or by whitefly transmission of the cloned progeny were unsuccessful, suggesting that additional factors are required for infection of the natural host. The significance of A. conyzoides as a reservoir host for the economically important geminivirus diseases is discussed.
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Differences in the subcellular localization of tobacco mosaic virus and cucumber mosaic virus movement proteins in infected and transgenic plants
More LessOur study reveals differences in the way that tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) movement proteins (MPs) partition with cellular components separated into four fractions from different aged leaves of infected and transgenic plants. Immunoblot analyses showed that TMV and CMV MPs associated predominantly with components in the cell wall fractions from leaves of transgenic plants. In infected tissue, highest levels of TMV MP were found in the organelle fractions from young and middle-aged leaves whereas most of the CMV MP was found in the detergent wash of the cell wall fraction. These results remained consistent even when plants were doubly infected with TMV and CMV. These results imply that MPs of plant viruses from different taxonomic groups differentially associate with subcellular components and that MP produced by a viral infection is targeted to additional subcellular destinations than MP produced in transgenic plants.
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Particle bombardment drastically increases the infectivity of cloned DNA of zucchini yellow mosaic potyvirus
More LessAn infectious full-length cDNA clone of the RNA genome of the potyvirus zucchini yellow mosaic virus (ZYMV) was constructed under the control of the cauliflower mosaic virus 35S promoter. All squash, cucumber, melon and watermelon plants inoculated with the cloned cDNA of ZYMV by particle bombardment become infected. Bombardment technology is 106-fold more effective than mechanical inoculation. Due to the great increase in efficiency, ineffective constructs now became infective (i.e. cDNA under the control of the 35S promoter without the NOS terminator; with an addition of 127 nucleotides at the 5′ end of the viral cDNA; uncapped transcripts), and the infectivity of capped-transcripts was maximized. Inoculation by particle bombardment produced visual symptoms rapidly (3–4 days), allowing the detection of viral coat protein and virions after 2 and 3 days in systemically infected leaves and inoculated cotyledons respectively.
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Replication slippage in the evolution of potyviruses
More LessRecently published evidence for sequence repetition in potyvirus genomes prompted us to analyse the published complete genome sequences and coat protein gene sequences of viruses of this family for evidence of replication slippage. Five of nine complete genomic sequences and 17 of 32 coat protein genes had significant sequence repetitions. Most of these were in coat protein genes, although the 5′ region of the turnip mosaic virus genome also showed evidence of slippage. The results suggest that replication slippage may be involved in the evolution of viruses, as well as prokaryotes and eukaryotes, and that slippage can occur in both RNA and DNA when it is being replicated.
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The nucleotide sequence of citrus leaf rugose virus RNA 1
S. W. Scott and Xin GeThe nucleotide sequence of citrus leaf rugose virus (CiLRV) RNA 1 consists of 3404 nucleotides and contains one open reading frame (ORF) which encodes a putative translation product of 1051 amino acids with a calculated M r of 118 339. Both the nucleotide sequence of CiLRV RNA 1 and its translated polypeptide share similarities with those of the RNA 1 of alfalfa mosaic virus. However, the relationship is not as close as that which exists between the polymerase signatures of the two viruses, which are found on RNA 2. This is the first report of the full-length sequence for the RNA 1 of an ilarvirus and completes the first sequence for an entire ilarvirus genome. If it is typical of members of the genus then, as has long been speculated, the genomic organization of ilarviruses is identical to that of other genera in the family Bromoviridae.
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