- Volume 73, Issue 11, 1992
Volume 73, Issue 11, 1992
- Articles
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- Animal
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Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus
More LessThe nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted M r of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5′ regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus poly-hedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.
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Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein
More LessThogoto (THO) virus is a tick-borne virus which shares morphological and genetic features with members of the Orthomyxoviridae family although the viral glycoprotein appears to be related to gp64 of baculoviruses. Characterization of THO virus was undertaken to clarify its taxonomic position. Purified virus preparations contained at least six virus-encoded polypeptides with apparent M r values ranging from 29K to 92K. A 75K polypeptide was identified as an envelope-associated glycoprotein by Triton X-100 and salt dissociation studies, and by proteolytic degradation of the exposed proteins of the virion. By the same criteria, the nucleoprotein and the matrix protein were identified as the 52K and 29K polypeptides, respectively. Immunofluorescence studies using monoclonal antibodies (MAbs) located the glycoprotein on the external cell membrane and the nucleoprotein in the nucleus of infected cells indicating that virus replication involved a nuclear phase. In addition, the virus displayed haemagglutination and haemolytic activities with an optimum at pH 6. These activities are functions of the viral glycoprotein since they were inhibited by antiglycoprotein MAbs. The data reported here support the notion that THO virus is a member of the Orthomyxoviridae family but that it should be classified in a group distinct from the other influenza viruses.
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Fusion of influenza virus particles with liposomes: requirement for cholesterol and virus receptors to allow fusion with and lysis of neutral but not of negatively charged liposomes
More LessInfluenza virus particles are able to fuse with liposomes composed of negatively charged or neutral phospholipids, as shown by using fluorochrome-labelled virions and fluorescence dequenching methods. Fusion with liposomes composed of only phosphatidylcholine (PC) was dependent on the presence of cholesterol (Chol), whereas fusion with liposomes containing negatively charged phospholipids, such as phosphatidylserine (PS), or of PC and phosphatidylethanolamine (PE) occurred in the absence of Chol. Fusion of influenza virions with PC:Chol liposomes was observed at pH 5.0, but not at pH 7.4, whereas a low degree of fusion with negatively charged liposomes or those containing PE was observed at pH 7.4. In addition, non-fusogenic influenza virions or HA0 influenza virions fused with PS- or PE-containing liposomes, especially at pH 5.0. Influenza virus particles were also able to induce the release of the fluorochrome calcein from negatively charged calcein-loaded liposomes at pH 5.0, as well as at pH 7.4, but failed to do so with PC:Chol liposomes. Lysis of PC:Chol by influenza virions was dependent on the presence of virus receptors, namely gangliosides (sialoglycolipids), and was observed only at pH 5.0. The results show that fusion of influenza virions with negatively charged or PE-containing liposomes does not reflect the biological activity of the virus needed for penetration and infection of living cells. On the other hand, fusion with PC:Chol liposomes is probably due to the activity of the viral fusion protein, the haemagglutinin glycoprotein.
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Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes
Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.
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Analysis of a 9.6 kb sequence from the 3′ end of canine coronavirus genomic RNA
More LessWe have analysed the organization of the 3′ end of the genomic RNA of canine coronavirus (CCV), a virus which has a close antigenic relationship to transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV) and feline infectious peritonitis virus (FIPV). Genomic RNA isolated from CCV strain Insavc-1-infected A72 cells was used to generate a cDNA library. Overlapping clones, spanning approximately 9.6 kb [from the 3′ end of the polymerase gene, 1b, to the poly(A) tail] were identified. Sequencing and subsequent analyses revealed 10 open reading frames (ORFs). Three of these code for the major coronavirus structural polypeptides S, M and N; a fourth codes for a small membrane protein, SM, a putative homologue of the IBV structural polypeptide 3c, and five code for polypeptides, designated 1b, 3a, 4, 7a and 7b, homologous to putative non-structural polypeptides encoded in the TGEV or FIPV genomes. An extra ORF which had not hitherto been identified in this antigenic group of coronaviruses was designated 3x. Pairwise alignment of these ORFs with their counterparts in TGEV, PRCV and FIPV revealed high levels of identity and highlighted the close relationship between the members of this group of viruses.
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Evidence of genomic variations between infectious pancreatic necrosis virus strains determined by restriction fragment profiles
More LessInfectious pancreatic necrosis virus (IPNV) is the aetiological agent of an important disease in hatchery-reared salmonid fish in North America, Europe and Japan. It belongs to the family Birnaviridae and shows a high degree of antigenic heterogeneity. However, genomic variations between the 10 identified serotypes have not yet been studied. In order to correlate genomic heterogeneity with the different serotypes, oligonucleotides were synthesized according to the published sequence of the Jasper strain (serotype A9). They were used as primers for the amplification of a 359 bp cDNA fragment of the viral genome using the polymerase chain reaction. Fragments amplified from 37 strains were digested with five different restriction enzymes. Restriction fragment profiles obtained on agarose gels showed heterogeneity not only between strains of different serotypes, but also among those belonging to serotype A1. A cluster analysis of the restriction patterns showed that IPNV strains can be divided into three major groups, corresponding approximately to serotypes A1, A2 and A3, and 10 subgroups which do not correlate with the serotyping of the strains.
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Bovine polyomavirus, a cell-transforming virus with tumorigenic potential
More LessThe early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.
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Analysis of splice sites in the early region of bovine polyomavirus: evidence for a unique pattern of large T mRNA splicing
More LessThe genetic organization of the early region of bovine polyomavirus (BPyV) was studied by analysis of the splice sites used in early mRNA maturation, using reverse transcription-polymerase chain reaction and DNA sequencing techniques. When compared to other polyomaviruses, the BPyV early region appears to have an uncommon organization. In the major early mRNA molecule two small intron sequences of 71 and 77 nucleotides, separated from one another by an 80 nucleotide exon sequence, were identified. Through splicing out both introns, a mRNA molecule is generated that contains an open reading frame with the capacity to encode 619 amino acids. Comparisons with the simian virus 40 large T antigen suggested that this mRNA molecule encodes the BPyV large T antigen. Remarkably, no mRNA product encoding a protein with a size comparable to that of the small t antigens of other polyomaviruses was detected. Another transcript was observed from which only the 77 nucleotide intron sequence had been removed, thereby creating a mRNA molecule with the capacity to encode only 45 amino acids. Whether this mRNA product represents a mature transcript which is translated in BPyV-infected cells or is an intermediate in the formation of the large T mRNA molecule is not known. Analysis of BPyV-specific early mRNA products isolated from BPyV-transformed murine cells revealed only the amplification product representing the putative large T antigen transcript.
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Nucleotide sequence of 21.8 kbp of variola major virus strain Harvey and comparison with vaccinia virus
More LessA 21.8 kbp region of the genome of variola major virus (strain Harvey), a virus that caused haemorrhagic-type smallpox, has been sequenced and shown to possess 96% nucleotide identity to the corresponding region of vaccinia virus, the smallpox vaccine. Overall the gene arrangement in the two viruses is highly similar and individual open reading frames (ORFs) display a high degree of amino acid identity, for instance 26 of the 32 variola virus ORFs have ≥90% identity with their vaccinia virus counterparts. A remarkable difference is the disruption of seven vaccinia virus ORFs into small fragments in variola virus. These include the variola virus homologue of vaccinia virus SalF2R, which encodes a protein related to C-type animal lectins, and SalF7L, which encodes an active 3β-hydroxysteroid dehydrogenase enzyme that contributes to vaccinia virus virulence. Upstream of the variola virus haemagglutinin gene there is a deletion of 1910 bp so that the equivalent of vaccinia virus gene SalF17R is truncated, and SalF16R, which shows amino acid similarity to the tumour necrosis factor receptor, is absent. The region sequenced includes the genes for thymidylate kinase and DNA ligase both of which are active in vaccinia virus and are highly conserved in variola virus. Other conserved ORFs with interesting homologies are those encoding profilin, superoxide dismutase and part of guanylate kinase. Two vaccinia virus genes encoding glycoproteins of the outer envelope of extracellular enveloped virus are also conserved in variola virus and this homology is likely to have contributed to the immunological protection which vaccinia virus evoked against smallpox. Lastly, there are multiple instances in which short oligonucleotide direct repeats flank a region absent from either variola or vaccinia virus.
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Nucleotide sequence analysis of a unique near-terminal region of the tumorigenic poxvirus, Shope fibroma virus
More LessShope fibroma virus (SFV), a tumorigenic poxvirus, has a DNA genome of approximately 160 kb. Previous DNA sequence analysis of SFV has been mainly limited to the terminal inverted repetitions (about 12 kb at each end of the genome) and immediately adjacent regions. We have sequenced a 4 kb fragment located approximately 20 kb from the right-terminal hairpin. Within this region three complete and two partial open reading frames (ORFs) have been identified. Each of the putative polypeptides has sequence similarity to one or more previously identified poxvirus or cellular proteins, with homology to protein kinases, erythrocyte ankyrin and a vaccinia virus virulence-related protein (ORF N1L). The potential significance of these gene products with regard to the phenotype of SFV is discussed.
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Assembly of conformation-dependent neutralizing domains on glycoprotein B of human cytomegalovirus
More LessWe analysed the antigenic properties of human cytomegalovirus (CMV) glycoprotein B (gB) by constructing a set of deletion derivatives lacking different portions of the carboxy terminus and reacting them with a panel of monoclonal antibodies with neutralizing activity. We found that two novel antigenic domains that bind neutralizing antibodies were assembled on truncated forms of gB, one in the aminoterminal half and one that spans the midregion of the molecule. Assembly of the conformation-dependent epitopes occurred independently of residues in the carboxy-terminal half of the molecule and did not depend on proteolytic cleavage of the molecule between amino acids 460 and 461. Ten antibodies recognized a derivative with 447 amino-terminal residues; their failure to recognize a derivative 411 residues long suggested that the amino acids required for assembly of these epitopes either were incorrectly folded, or had been totally or partially deleted in this derivative. Epitopes for three antibodies with complement-independent neutralizing activity were assembled when amino acids from the midregion of gB between residues 447 and 476 were present. Two other antigenic domains were formed by the addition of residues 476 to 618 and 619 to 645 from the carboxy-terminal half of gB. Our results underscore the importance of conformation in the antigenic structure and functional properties of both the amino- and carboxy-terminal portions of gB.
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The lower matrix protein pp65 is the principal viral antigen present in peripheral blood leukocytes during an active cytomegalovirus infection
During an active infection with human cytomegalovirus (HCMV), viral antigen is consistently present in peripheral blood leukocytes. Two monoclonal antibodies (MAbs), CMV-C10 and CMV-C11, are commonly used in the HCMV antigenaemia assay to detect these cells in the peripheral blood of patients suspected of having an active HCMV infection. We demonstrate that the viral antigen detected by these MAbs is the viral structural protein pp65 and not an immediate early antigen as previously reported. Furthermore, significantly fewer leukocytes were found to be positive with MAbs specific for immediate early antigens.
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Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?
A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.
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Characterization and transcript mapping of a bovine herpesvirus type 1 gene encoding a polypeptide homologous to the herpes simplex virus type 1 major tegument proteins VP13/14
More LessUsing in vitro translation of hybrid-selected mRNA, we have previously shown that bovine herpesvirus type 1 HindIII fragment M encodes an abundant 94K polypeptide. Using immunoprecipitation and sequencing analyses, it has now been shown that the polypeptide is related to the major tegument protein VP8 and is homologous to the herpes simplex virus type 1 major tegument proteins VP13/14. The sequence of the VP8 gene (field isolate 34) is reported and compared to published data. Several differences between the sequences were detected, resulting particularly from base insertions/deletions generating three major frameshifts affecting an area of 87 amino acid residues of the encoded protein. In addition, sequence comparison revealed 29 single base alterations, excluding frameshift regions, producing 17 amino acid substitutions. Overall, 14.1% of the deduced amino acid sequences were divergent. We have also established that the last 152 nucleotides of the previously reported sequence correspond to the sequence of the minus not the sense strand. Finally, we report that the 4.4 kb transcript of the VP8 gene is initiated 39 nucleotides upstream from the translation start codon.
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Cyclic AMP-mediated inhibition of vesicular stomatitis virus and herpes simplex virus replication in mouse macrophage-like cells
More LessIn this study, we have analysed the effects of cAMP inducers on the multiplication of vesicular stomatis virus (VSV) and herpes simplex virus type 1 (HSV-1) in mouse macrophage-like cells. The addition of dibutyryl cAMP (dB-cAMP) or cholera toxin to resting peritoneal macrophages aged in vitro or P388D1 cells resulted in a 10- to 100-fold reduction of VSV yield compared to control cultures. In contrast, no cAMP-dependent inhibition was found in VSV-infected L929 cells. In macrophage-like cells, the dB-cAMP-induced antiviral state was not inhibited by antibodies to interferon (IFN)-α/β and did not correlate with any increase in the intracellular levels of 2–5 oligo(A) synthetase. Dibutyryl cAMP did not inhibit virus yields in mouse macrophages infected with encephalomyocarditis virus. In P388D1 cells, the addition of dB-cAMP resulted in an approximately 10-fold inhibition of HSV-1 replication with respect to control cultures, as evaluated both by TCID50 and plaque assays on Vero cells. Dibutyryl cAMP did not affect VSV binding or entry into mouse macrophages and the cAMP-mediated anti-VSV state was significantly reduced by inhibitors of protein kinase C (i.e. staurosporine and H7). These data suggest that macrophages may acquire resistance to infection by VSV and HSV-1 after treatment with cAMP inducers. This cAMP-mediated antiviral activity does not depend on the modulation of the endogenous IFN system, suggesting that macrophages exhibit multiple resistance mechanisms (i.e. IFN-dependent and IFN-independent) to maintain their intrinsic antiviral activity.
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Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0
More LessDominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.
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Nucleotide sequence analysis of genes encoding glycoproteins D and J in simian herpes B virus
More LessThe gene encoding glycoprotein D (gD) of simian herpes B virus (SHBV) was identified by hybridization with the gD gene of herpes simplex virus type 1 (HSV-1). The gene probe bound to a 2.6 kbp SalI-EcoRI fragment of SHBV DNA, which was cloned into a plasmid vector. The nucleotide sequence of the SHBV DNA fragment was determined. Two complete and one partial open reading frames (ORFs) were found. The nucleotide sequences of the two complete ORFs are 57% and 69% identical to HSV-1 genes US5 (encoding gJ) and US6 (encoding gD), respectively. The partial ORF showed 64% similarity with HSV-1 US7 (encoding gI). The SHBV gD gene revealed many features which are also found in the gD homologues of other herpesviruses. The positions of cysteine residues and receptor-binding sites for the predicted protein are shown to be highly conserved.
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Human sera from varicella-zoster virus (VZV) infections cross-react with human T cell leukaemia virus type 1 (HTLV-1): common epitopes in VZV gene 22 protein and HTLV-1 p19 gag protein
Twenty-nine of 100 sera from patients recently infected with varicella-zoster virus (VZV) were found to cross-react with human T cell leukaemia virus type 1 (HTLV-1) antigen in the particle agglutination (PA) assay using HTLV-1 antigen-coated gelatin particles. Anti-VZV IgM antibodies were shown to be responsible for this cross-reactivity. Western blot analysis revealed that PA-positive anti-VZV sera reacted with the HTLV-1 gag p19 protein in HTLV-1-infected cells and recombinant p19 protein produced in Escherichia coli. By using a truncated p19, the cross-reactive region was located to the C-terminal 17 amino acids of p19. One oligopeptide derived from the C terminus, PQIP-PPYVEPT (amino acids 115 to 125), was capable of inhibiting PA, suggesting that this peptide carries the cross-reactive epitope. A homologous sequence was found in the VZV gene 22 protein by database analysis, and the oligopeptide TNIPPPLALLR (amino acids 1330 to 1340) had the ability to inhibit PA. These findings suggest that some IgM antibodies against the VZV gene 22 protein produced in the early phase of VZV infection are cross-reactive with the HTLV-1 gag p19 protein because they recognize an antigenic determinant containing an IPPP tetrapeptide.
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Open reading frames 1 and 2 of adenovirus region E4 are conserved between human serotypes 2 and 5
More LessThe E4 region of human adenovirus type 2 is predicted to encode seven proteins as judged from its nucleotide sequence and the pattern of differential splicing of its transcript. Two of the open reading frames (ORFs), ORF1 and ORF2, had been identified as being disrupted in the recently published sequence of the related serotype 5 virus. These ORFs were resequenced and found to be intact in the wt300 strain of adenovirus type 5.
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Adeno-associated virus type 2-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) replication: involvement of p78rep/p68rep and the HIV-1 long terminal repeat
More LessMicroinjection of wild-type adeno-associated virus type 2 (AAV-2) DNA and infectious human immunodeficiency virus type 1 (HIV-1) proviral DNA into the nuclei of human epithelioid SW480 cells leads to specific inhibition of HIV-1 replication. Mutational analysis of the AAV genome showed that this negative interference can be assigned to a functional AAV-2 rep gene. Moreover, the p78rep/p68rep proteins are sufficient for the anti-HIV-1 effects. The rep gene also inhibits the expression of a chloramphenicol acetyltransferase (CAT) gene driven by the U3/R portion of the HIV-1 long terminal repeat (LTR) in the absence of tat expression. This suggests that the U3/R portion of HIV-1 contains elements responsible for the AAV-2 rep-mediated inhibition of HIV-1 LTR-driven CAT gene expression and, probably, also of HIV-1 replication. The results add support for the general significance of AAV-2 and specifically the rep gene as tools for down-regulating heterologous gene expression.
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Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus
Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.
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Sequences responsible for efficient replication of simian immunodeficiency virus SIVMND in cells of the monocyte/macrophage lineage
More LessWe determined the susceptibility of monocytic cell lines to infection with viral strains derived from two infectious clones of simian immunodeficiency virus isolated from a mandrill. One of the strains, which replicates poorly in T cell lines, was found to grow more rapidly than the other in these cells. The viral determinant for this property was genetically mapped within the env gene encoding a surface protein. Six amino acid substitutions identified appeared to be located outside of the domains corresponding to human immunodeficiency virus type 1 env functional domains such as the CD4-binding and V3 loop regions.
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Host range of Rous sarcoma virus pseudotype RSV(HPRS-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated J
More LessThe host ranges of the Rous sarcoma virus (RSV) pseudotype RSV(HPRS-103) of a novel avian leukosis virus (ALV), strain HPRS-103, and representative RSV pseudotypes of subgroups A to F, have been determined in embryo fibroblasts from 12 avian species. Domestic fowl, red jungle fowl, Sonnerat’s jungle fowl and turkey were susceptible to infection by RSV(HPRS-103); ring-necked pheasant, Japanese green pheasant, golden pheasant, Japanese quail, guinea-fowl, Peking duck, Muscovy duck and goose were resistant. The host range pattern of RSV(HPRS-103) differs from those of viruses of subgroups A to G and I, and provides support for placing the HPRS-103 strain of ALV in a new envelope subgroup, designated J.
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Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies
More LessAnti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-shocked cells, but were first detected in infected cells approximately 32 h post-infection. PEPSCAN epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (9RNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (6KKAR9) and spans a possible second site for the initiation of synthesis of core protein (12PFN↓MLKR18).
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Genomic variability in the preS1 region and determination of routes of transmission of hepatitis B virus
On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.
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The pathogenicity of two porcine rotaviruses differing in their in vitro growth characteristics and genes 4
More LessThe pathogenicity of two rotavirus variants, 4F and 4S, obtained following adaptation to cell culture of rotavirus from a diarrhoeic pig in China, was compared by serial passage in 24 gnotobiotic piglets. The rotavirus variants have markedly different growth characteristics in vitro, and their genome profiles differ only in the relative migration of genes 4. Both cell culture-grown variants replicated to an equal extent in gnotobiotic piglets and neither caused disease, although weight gain was slightly affected in piglets inoculated with the 4F variant. During five serial pig-to-pig passages, variant 4F became highly pathogenic at the fourth and fifth passages, causing severe diarrhoea and weight loss, and premature death in two animals. Piglets inoculated with rotavirus variant 4S remained healthy during all passages although weight gain was slightly affected. Mean duration and peak infectivity titres of virus shedding were similar for both variants. Thus, variant 4F, which grew slowly and produced small plaques in vitro and had the faster migrating gene 4, was pathogenic in pigs, whereas variant 4S was apathogenic.
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Inhibition of rotavirus in vitro transcription by optimal concentrations of monoclonal antibodies specific for rotavirus VP6
More LessThree monoclonal antibodies (MAbs) obtained from inoculation of mice with either a serotype 1 human rotavirus or rotavirus SA11 (serotype 3) inhibited the in vitro transcription of rotavirus SA11. Two of the MAbs exhibited a biphasic inhibitory response. Removal of antibody from MAb preparations by adsorption with Sepharose-Protein G reduced the inhibitory activity completely for all three MAb preparations. Analysis by radioimmunoprecipitation and Western blotting indicated that all three MAbs reacted with VP6. All MAbs also reacted with four group A rotavirus serotypes by ELISA, but did not cross-react with reovirus type 1, poliovirus type 2 or MA-104 cell lysates. Transcription of four rotavirus serotypes as well as epizootic diarrhoea of infant mice rotavirus was inhibited when tested with two of the MAbs. Transcription of both purified single-shelled virus and purified heat-activated double-shelled SA11 rotavirus was inhibited by purified MAb. Our results indicate that these MAbs can be used effectively to study the events associated with rotavirus transcription.
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Sequence of genome segment 9 of bluetongue virus (serotype 1, South Africa) and expression analysis demonstrating that different forms of VP6 are derived from initiation of protein synthesis at two distinct sites
More LessBluetongue virus (BTV) VP6 is often resolved into two closely migrating bands by SDS-PAGE (VP6 and VP6a). RNA segment 9 of BTV-serotype 1 South Africa (encoding VP6) has been cloned as cDNA, and the complete sequence has been determined. Expression of this clone both in vitro and in tissue culture produced the same polypeptide doublet as seen previously in extracts from BTV-infected cells. Modification of the cDNA, including the removal of the first initiation codon, demonstrated that the two forms of VP6 are derived from initiation of protein synthesis at two distinct sites and not by post-translational modification.
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Demonstration of scrapie strain diversity in infected PC12 cells
Scrapie strain replication in the nerve growth factor-induced, differentiated PC12 cell culture system was examined. Differences in replication between mouse-derived agents were demonstrated, with the 139A scrapie strain yielding 100- to 1000-fold higher levels of infectivity than the ME7 scrapie strain. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Studies on the neurotransmitters in infected PC12 cells demonstrated that the adrenergic pathway was unchanged but the cholinergic pathway was altered. Furthermore, the degree of alteration correlated with the level of scrapie strain replication. Comparison of infectivity titres and enzymatic changes in ME7-infected PC12 cells with those in Chandler agent-infected mouse neuroblastoma cells suggests that the significant changes in neurotransmitter levels in cultures exhibiting low titres of infectivity involve factors in addition to strain replication. The variation in the range of scrapie strain replication in PC12 cells is discussed in relationship to species barrier, cell targeting, genetic susceptibility and species strain specificity. These studies further emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and in addition support the concept of variation among scrapie strains.
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A viroid from Nematanthus wettsteinii plants closely related to the Columnea latent viroid
More LessA viroid was isolated from symptomless Nematanthus wettsteinii plants using the return-PAGE method for analysis of low M r nucleic acids. The RNA was transmitted to tomato, three cultivars of potato, and Scopolia sinensis plants by mechanical inoculation or by grafting. Infected solanaceous plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). The Nematanthus viroid consists of 372 nucleotides, 214 G+C, 158 A+U, with a G+C/A+U ratio of 1.35. One of seven cDNA clones showed a sequence heterogeneity (G to A) at position 73. The most stable secondary structure of this viroid has 78 G:C, 37 A:U and 11 G:U base pairs with a minimum free energy of -456.9 kJ. The viroid is closely related to the 370 nucleotide Columnea latent viroid. The Nematanthus viroid possesses regions of 100% sequence identity with six viroids belonging to the PSTVd and apple scar skin viroid groups. The viroid also replicated in tomato plants when mixed with PSTVd. Tomato plants were cross-protected against PSTVd when preinfected with the viroid from N. wettsteinii.
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Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells
More LessThe coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.
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The complete nucleotide sequence of turnip mosaic potyvirus RNA
More LessThe complete RNA genome of turnip mosaic potyvirus (TuMV) was amplified by seven consecutive reverse transcriptase-polymerase chain reactions and cloned into pUC9. The viral RNA is 9830 nucleotides long and contains a single open reading frame (ORF) of 9489 bases encoding a large polyprotein of 3863 amino acids with a calculated M r of 358000. The non-coding region (NCR) preceding the ORF is 129 nucleotides long and has a high AU content (70%). Its predicted secondary structure is characterized by a hairpin loop with a free energy loss of -69.9 kJ/mol. The termination codon is followed by an AU-rich NCR of 209 bases, excluding the poly(A) tail. Seven potential nuclear inclusion a proteinase (NIa-Pro) recognition heptapeptides are found in the polyprotein. Their sequences agree with consensus potyviral NIa-Pro cleavage sequences except for that at the 6K-VPg site, which is characterized by a glutamic acid residue preceding the hydrolysed peptide bond. The TuMV proteins are similar to their corresponding potyviral proteins.
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The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments
More LessThe complete sequence of the tomato spotted wilt virus (TSWV) M RNA segment has been determined. The RNA is 4821 nucleotides long and has an ambisense coding strategy similar to that of the S RNA segment. The M RNA segment contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted size of 33.6K, and one in the viral complementary sense which encodes the precursor to the G1 and G2 glycoproteins, with a predicted size of 127.4K. Both ORFs are expressed via the synthesis of subgenomic mRNAs that possibly terminate at a stable hairpin structure, located in the intergenic region. The precursor for the glycoproteins contains a sequence motif (RGD) which is characteristic of cellular attachment domains. Significant sequence homology was found between the G1 glycoproteins of members of the genus Bunyavirus and a corresponding region in the glycoprotein precursor of TSWV, indicating a close evolutionary relationship between these viruses. With the elucidation of the M RNA sequence, the complete nucleotide sequence of TSWV has been determined. TSWV represents the first member of the Bunyaviridae shown to contain two ambisense RNA segments.
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Widely separated sequence elements within cucumber mosaic virus satellites contribute to their ability to induce lethal tomato necrosis
Gusui Wu and J. M. KaperTo determine the structural requirements for cucumber mosaic virus (CMV) satellites to elicit lethal tomato necrosis, three satellite variants D, S and Y were used in the construction and cloning of chimeric cDNAs. D and S are necrogenic and non-necrogenic ‘prototype’ variants, respectively, and Y possesses the 3′ conserved necrosis-determining region but does not cause lethal tomato necrosis. Its 5′ half harbours an insertion/deletion region that results in a molecule about 30 nucleotides longer than other variants. Tomato bioassays were conducted with RNA transcripts of all six chimeric combinations of the 5′ and 3′ halves of the three satellite variants divided by a common restriction site, as well as with a mutated chimera. None of the chimeras containing the 5′ half of Y induced lethal necrosis in tomato even when their 3′ halves were that of the D variant with the conserved necrogenic element. Chimeras with the 3′ half of Y elicited only partial or restricted necrosis which was much less severe than that induced by prototype variant D, and often was not lethal. Site-directed mutation of a single nucleotide in proximity to the necrogenic element of such a chimera containing the 3′ half of Y restored much lethal necrogenicity. The results revealed the presence of structural elements in CMV satellite variant Y that modulate or even suppress the expression of the 3′ conserved necrosis-determining element. They indicate that in CMV satellites widely separated sequence elements constituting a three-dimensional requirement are responsible for eliciting lethal necrosis in tomato.
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Immunodetection of grapevine fanleaf virus satellite RNA-encoded protein in infected Chenopodium quinoa
More LessAn antiserum was raised against a fusion protein containing the C-terminal half of the protein (P3) encoded by the satellite RNA of grapevine fanleaf virus (GFLV; F13 isolate) and the N-terminal portion of the CI repressor of phage λ. This antiserum specifically recognized P3 synthesized in the in vitro wheatgerm translation system and also in infected Chenopodium quinoa plants. In these plants, the amount of virus increased for 10 days, then remained constant for up to 21 days, whereas P3 was detected transiently, reaching its maximum on day 10.
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Intracellular distribution of the 126K/183K and capsid proteins in cells infected by some tobamoviruses
Pritam Das and V. HariLeaves of plants infected by tobacco mosaic virus (TMV) strain U1, TMV strain M, tomato mosaic virus strain Dahlemense and tobacco mild green mosaic virus strain U2 were examined for the presence and intracellular distribution of their capsid and 126K/183K proteins by immunoblotting and immunogold electron microscopy. The bulk of the capsid protein was found in the virus bundles (crystals), although small amounts were found in the chloroplasts and nuclei of cells infected by some of these tobamo-viruses. The 126K protein of TMV-U1 and -M was localized in the X-bodies, whereas in cells infected by the other two viruses which induce no X-bodies, the 126K protein was found to be associated with virus bundles.
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Diffraction studies of the particles of two closteroviruses: heracleum latent virus and heracleum virus 6
More LessX-ray diffraction from oriented specimens of purified preparations of particles of heracleum latent clostero-virus (HLV) showed that they have a helical arrangement of protein subunits, and that the structure repeats in five helical turns in which there are 5q ± 1 protein subunits, where q is an integer. The pitch of the helix was estimated to be 3.26 (±0.10) nm. Optical diffraction patterns from electron micrographs of HLV particles give an estimated pitch of 3.3 (±0.2) nm and show that the number of subunits in the repeat period is 5q-1, where q has a value of 8 or 9. Optical diffraction from electron micrographs of particles of a second closterovirus, heracleum virus 6, shows that they too have a helical structure which repeats in five turns, in which there are 5q ± 1 protein subunits. The estimated pitch of the primary helix is 3.6 (± 0.2) nm, and the estimate of q is 9.
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