- Volume 72, Issue 6, 1991
Volume 72, Issue 6, 1991
- Animal
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Biophysical and biochemical properties of an unusual birnavirus pathogenic for rotifers
More LessA cytoplasmic dsRNA virus, rotifer birnavirus (RBV), has recently been isolated from the rotifer Brachionus plicatilis and is associated with a high mortality rate. Histologically, the viral lesions consist of characteristic inclusions, particularly amorphous dense bodies containing occluded particles. Purified virions are about 59 nm in diameter, single-shelled and display four capsomers per edge. The purified virions have a buoyant density of 1.290 (full particles) and 1.250 (empty particles) in CsCl gradients. Four major structural polypeptides of M rs 60K, 52K, 33K and 27K were detected by SDS-PAGE. The genome is composed of two linear segments of dsRNA with M rs of 2.45 × 106 and 2.31 × 106; additionally, small circular ssRNA molecules were detected by electrophoresis in over-loaded agarose gels, but their significance is currently unknown. Except for this last feature and the structural instability of purified virions under freeze storage, all the other biochemical and biophysical characters indicate that RBV is a member of the Birnaviridae family with, for the moment, a unique position in this group.
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The complete sequence of the group-specific antigen, VP7, of African horsesickness disease virus serotype 4 reveals a close relationship to bluetongue virus
More LessThe complete sequence of the S7 RNA that codes for the major group-specific coat protein, VP7, of African horsesickness virus serotype 4 (AHSV-4) was determined from cDNA analyses and found to be 1179 nucleotides in length. One single open reading frame of 353 codons was observed defining a protein of M r 38107 with a net charge of -1.5 at neutral pH. Comparison of the AHSV-4 VP7 sequence with that of bluetongue virus serotype 10 revealed an overall similarity of 44%, with the amino- and carboxy-terminal regions exhibiting the greatest levels of homology. In addition, potential secondary structures of the terminal sequences of the S7 RNA segments of AHSV-4 and BTV serotypes 10, 13 and 17 are presented.
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Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system
The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.
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Susceptibility of cultured human trophoblasts to infection with human immunodeficiency virus type 1
Primary cultures of essentially pure human term trophoblasts were studied to determine their ability to support the expression of complete proviral clones of human immunodeficiency virus (HIV) and their permissiveness to this virus. Transient expression of molecular clones derived from two biologically distinct strains, BRU and NDK, resulted in the release of comparable amounts of infectious virions, which were rescued by cocultivation with permissive CEM-SS cells. Trophoblasts were inoculated with three HIV-1 isolates, RF, 3B and NDK, which differ in their cytopathogenicity on T lymphoblastoid cells. Infection of cells by all three strains was demonstrated by the presence of virus-specific proteins in the trophoblasts and the detection of virus gag gene-related DNA sequences by the polymerase chain reaction (PCR), but cells were more susceptible to infection with the RF and NDK strains than with the 3B strain. The virus was readily transmitted to the CEM-SS cells with simultaneous formation of syncytia between the two cell types. Flow cytometry and direct radioimmunoassay revealed no trace of the CD4 receptor on the surface of the cultured trophoblasts and CD4 mRNA could not be detected by Northern blot hybridization, although a minimal amount of CD4-associated mRNA was detected by PCR. Our data suggest that infection of trophoblasts occurs independently of the pathway mediated by CD4.
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Characterization of human antibody-binding sites on the external envelope of human immunodeficiency virus type 2
More LessAntibody-reactive peptide scanning (Pepscan) using overlapping nonapeptides and human sera as probes allows the identification of amino acids contributing significantly to antigen-antibody interaction. Five-hundred and two overlapping nonapeptides derived from the human immunodeficiency virus type 2 strain Rod (HIV-2Rod) external envelope glycoprotein gp125 were synthesized to serve as probes for reactivity with eight sera of HIV-2-infected individuals. Fifteen antibody-binding regions were identified, among which two amino-terminal regions [E3, amino acids (aa) 118 to 132; E4, aa 125 to 141] and four carboxy-terminal regions (E11, aa 303 to 324; E12, aa 340 to 358; E14, aa 436 to 452; E15, aa 486 to 507) were the most antigenic. The amino acids in binding sites E3 and E4 were highly variable among simian immunodeficiency virus (SIV), HIV-2 and HIV-1. The antibody-binding domains E14 and E15 were highly conserved among HIV-2 strains (94% and 86% identity of HIV-2Rod to HIV-2Isy and HIV-2NIHZ, respectively). Both domains had more amino acids in common with SIV (88% for E14, 64% for E15) than with HIV-1 (41% for E14, 45% for E15). Epidemiological studies revealed that the sera of African HIV-2-infected individuals bound the E11 and E15 peptides best (31%, 8/26). The sera of African HIV-1-infected individuals showed significant levels of cross-reactivity to the HIV-2 peptides, especially to the E15 peptide, whereas the sera of European HIV-1-infected individuals showed only moderate levels of cross-reactivity. If peptides covering the E15 epitope were used, African HIV-2-positive sera showed only a low level of cross-reactivity (4%) to E15 of HIV-1 and SIV. African HIV-1-positive sera bound the HIV-1 E15 peptide best (81%, 52/64), but showed high levels of cross-reactivity to SIV E15 (17%, 11/64) and HIV-2 E15 (25%, 16/64). European HIV-1-positive sera showed a high level of reactivity to HIV-1 E15 (91%, 50/55) and a low level of cross-reactivity to the HIV-2 (2%, 1/55), and none to the SIV E15 (0/55) peptide. These results indicate that the immunodominant regions of the HIV-2 external envelope (E11 and E15) align with the most immunodominant regions of HIV-1.
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Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins
Site-directed mutagenesis was used to study the biological significance of a disulphide bridge and two N-linked oligosaccharides in the CD4-binding region of the envelope glycoproteins of human immunodeficiency virus type 1. Mutagenesis was performed in a phage M13 system at sites corresponding to the cysteine residue (amino acid 402) and the asparagine residues (390 and 447) of the env gene. The mutated env gene was inserted into a recombinant vaccinia virus under the control of the vaccinia virus 7.5K promoter and the expression of mutated env proteins was analysed by SDS-PAGE, a conventional indirect immunofluorescence assay and by a fluorescence-activated cell sorter. Cysteine 402 was found to be essential for the specific cleavage of gp160 into gp120 and gp41, and for intracellular transport of the protein to the cell surface. CD4-binding and syncytium formation assays demonstrated that the disulphide bridge of cysteine 402 stabilized a conformation essential for receptor binding as well as syncytium formation by CD4+ cells. No altered biological activity compared to that of the wild-type proteins could be detected for the mutant proteins lacking the N-glycosylation sites. These data show that the two conserved glycans attached to asparagine residues 390 and 447 do not play any active role in the formation of the disulphide bridge involving cysteine 402 or in the maintenance of an active conformation of the protein, despite their location within the functionally important CD4-binding region.
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Alterations in neurotransmitter-related enzyme activity in scrapie-infected PC12 cells
More LessEnzyme activities associated with the neurotransmitter pathways in nerve growth factor-treated, 139A scrapie strain-infected PC12 cells were examined. Since these cells show no morphological alterations during the time of agent replication, any scrapie-induced effects would have to be associated with non-vital cellular functions. When compared to controls, infection with the 139A scrapie strain resulted in decreased activity of the cholinergic pathway-related enzymes, choline acetyltransferase and acetylcholinesterase. However, the adrenergic pathway was unaffected by scrapie infection as evidenced by unaltered tyrosine hydroxylase activity, the putative rate-limiting enzyme in the synthesis of catecholamines. The effects of the 139A scrapie strain on the cholinergic system appeared to be dose-dependent and were first detected prior to the detection of scrapie agent replication in these cells. Furthermore, the altered enzymic activities observed were not the result of contaminating material in the scrapie brain homogenate because similar results were obtained when partially purified scrapie preparations were used as the inoculum. These scrapie agent-induced alterations in specific neuronal properties suggest a mechanism for the clinical manifestations observed in scrapie and perhaps other related central nervous system disorders.
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Restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene and their association with susceptibility to natural scrapie in British sheep
More LessWe have investigated the correlation between restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene and the incidence of natural scrapie in British sheep during the period from July 1988 to November 1990. Sixty percent of the scrapie-positive animals studied were homozygous for a 6.8 kb EcoRI fragment (e1) and a further 26% carried e1 as heterozygotes. This fragment is linked to susceptibility to experimental scrapie in a closed flock of Cheviot sheep. Twelve percent of cases were found to be homozygous for a 4.4 kb EcoRI fragment (e3) which in the Cheviot flock has been linked to relative resistance to scrapie. A third EcoRI fragment of 5.2 kb (e2) has also been found but is relatively rare and has not yet been associated with scrapie susceptibility. Four sets of flocks affected by natural outbreaks of scrapie divided into two groups. In three of these flocks, all scrapie cases carried e1 with high frequencies of e1e1 homozygotes. In the fourth, there were no e1e1 scrapie cases; all scrapie sheep carried e3 in approximately equal numbers of heterozygotes and homozygotes.
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Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins
More LessThe immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.
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Regulated M1 mRNA splicing in influenza virus-infected cells
More LessInfluenza virus RNA segment 7 generates three poly(A)+ RNAs, M1 mRNA, M2 mRNA and mRNA3, the last of which has almost no coding capacity; M2 mRNA and mRNA3 derive from M1 mRNA by removal of a single intron. The kinetics of M1 and M2 mRNA accumulation in the cytoplasm of productively infected cells were studied by means of a quantitative RNA protection assay; the ratio of M2 mRNA to M1 mRNA increased 2.7-fold during the course of infection. To analyse the basis for this change, the kinetics of M1 and M2 mRNA synthesis and nuclear accumulation, their stability and nucleocytoplasmic transport were studied. Under the experimental conditions used, the synthesis of segment 7-specific RNA showed a peak at 4 h post-infection and continued later at a slower rate. The half-lives of M1 and M2 mRNAs were indistinguishable (2.73 h for M1 mRNA and 2.70 h for M2 mRNA) and the kinetics of nucleocytoplasmic transport in vivo or in vitro showed no preference for either mRNA early or late in infection. Consequently, regulation at the level of mRNA splicing is proposed. Using the mRNA synthesis and stability data, a simulation was performed to predict the change in splicing efficiency required to account for the mRNA accumulation results. The best fit was obtained when splicing efficiency changed about 20 times during a period in which viral gene expression was maximal.
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High level transient expression of the murine coronavirus haemagglutinin-esterase
More LessWe have expressed the murine coronavirus haemagglutinin-esterase protein in a vaccinia virus/T7 RNA polymerase system. The levels of expression observed are significantly higher than those found in virus-infected cells. The expressed protein has both receptor-destroying (esterase) and receptor-binding (haemad-sorption) activities. The use of this system will greatly facilitate analysis of the structure-function relationships of this protein.
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Acquired immunity of A/J mice to mouse hepatitis virus 3 infection: dependence on interferon-γ synthesis and macrophage sensitivity to interferon-γ
More LessCoronavirus-free A/J mice (A/J-), in contrast to those naturally infected with coronavirus (A/J+), were shown to be susceptible to experimental infection with our strain of mouse hepatitis virus 3 (MHV3). A/J-mice experimentally hyperimmunized with inactivated MHV3 (A/Ji) became resistant to challenge with this virus. BALB/c mice free of (BALB/c-) or naturally infected with (BALB/c+) coronavirus, or hyperimmunized with inactivated MHV3 (BALB/ci), were always fully susceptible. All susceptible mice developed an acute hepatitis with a high virus titre in the tissues. Resistance mice developed a mile disease in which the low virus titres detected in the tissues were cleared. After infection, interferon (IFN)-γ synthesis in A/J-mice was lower than that in A/J+ and A/Ji mice; IFN-γ synthesis was very high in BALB/c+ and BALB/ci mice, but low in BALB/c-mice. Studies of the anti-MHV3 effect induced in macrophages in vitro showed that only IFN-γ-activated A/J mouse macrophages were able to restrict partially the growth of MHV3, regardless of whether the animals had been immunized. The effect occurred only when the cells were activated with IFN-γ before virus infection. The results indicate that the resistance of A/J mice to our strain of MHV3 is not natural but is acquired after immunization, and that the mechanism involved is dependent on T cell activity, IFN-γ production and the sensitivity of macrophages to IFN-γ.
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Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions
More LessThe fusion activity of flaviviruses [tick-borne encephalitis (TBE) virus and Japanese encephalitis virus] was assessed by inducing fusion from without of C6/36 mosquito cells with purified virus preparations. Membrane fusion and polykaryocyte formation was observed only after incubating the viruses at acidic pH. Two groups of monoclonal antibodies reacting with distinct non-overlapping antigenic domains on the TBE virus protein E inhibited fusion from without. One of these domains contains the most highly conserved and putative fusion-active sequence of the flavivirus protein E. Of five TBE virus monoclonal antibody escape mutants, each defined by a single amino acid substitution in the envelope protein E, one revealed a reduced fusion activity and another one a lower pH threshold. TBE virus grown in the presence of ammonium chloride as well as Langat virus purified from the supernatant of infected chick embryo cells contained the precursor of protein M (prM) rather than M itself. These ‘immature’ virions did not cause fusion from without, suggesting that the proteolytic processing of prM may be necessary for the generation of fusion-competent virions.
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Characterization of yellow fever virus proteins E and NS1 expressed in Vero and Spodoptera frugiperda cells
More LessThe cDNA encoding the E and NS1 proteins of the yellow fever virus (YFV) was expressed in Spodoptera frugiperda cells via the recombinant baculovirus Ac-E. NS1 as a gp100 precursor which was cleaved to generate the recombinant proteins E and NS1 similar in size, folding and antigenicity to the authentic ones. Recombinant protein E exhibited immunodominant epitopes as judged by its reactivity with YFV-neutralizing MAbs. Using the Triton X-114 phase separation system, authentic and recombinant E proteins as well as the gp100 precursor exhibited hydrophobic properties similar to those of integral membrane proteins. Recombinant protein E was found neither in the extracellular medium nor on the cell surface, suggesting that it did not migrate within the secretory pathway of insect cells. Analysis of protein NS1 expressed in primate and insect cells revealed that the newly synthesized 48K NS1 glycoprotein was converted to a heat-labile gp72 homo-oligomeric form. This phenomenon did not require the presence of carbohydrate groups. Using the Triton X-114 phase separation system, the oligomeric form of NS1 was shown to be associated with cellular membranes although it appeared less hydrophobic than protein E and gp100. A small fraction of YFV NS1 oligomers were transported throughout the secretory pathway to be shed into the extracellular medium of primate cells. YFV NS1 oligomers migrated from the endoplasmic reticulum to the Golgi complex, whereas their N-oligosaccharides of the high-mannose type are processed to the complex-mannose type. Protein NS1 expressed by recombinant baculovirus-infected insect cells was not found in the extracellular medium but associated with the plasma membrane of the cells. Two recombinant NS1 forms were detected in insect cells: a major one with an apparent M r of 48K and a minor one of 47K in which N-linked glycans were probably processed to a trimannosyl core without further elongation. Thus, it appears that the transport strategy as well as the N-glycosylation of NS1 in insect cells infected with recombinant baculovirus were different from those of the NS1 in primate cells infected with YFV.
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Use of human papillomavirus type 11 virions in an ELISA to detect specific antibodies in humans with condylomata acuminata
More LessHuman papillomavirus types 6 and 11 (HPV-6 and HPV-11) are the major aetiological agents of condylomata acuminata. Serological studies of this disease have been difficult to perform and interpret because native, type-specific antigens have not been available. In particular, since these viruses have not been propagated in vitro and sufficient quantities of virions are not present in lesions, virus particles have been difficult to obtain. In the present study, we used HPV-11 particles, obtained from human tumours produced in athymic mice, as antigen in an ELISA to compare antibody responses between 46 patients with biopsy-proven condylomata acuminata and 44 controls. The median [interquartile range] of the absorbance values for the condylomata acuminata and the control groups were respectively 0.324 [0.183, 1.029] and 0.118 [0.047, 0.286] (P=0.001). Thirty-three per cent of the absorbance values in the condylomata acuminata group were higher than any of those of the control group. Sera from patients whose biopsies contained the papillomavirus common antigen were more reactive than sera from patients whose biopsies did not contain it (P=0.0014). This study demonstrates the presence of specific antibodies directed at native HPV-11 viral particles in the sera of patients with condylomata acuminata, and describes a test which can be used in future serological studies of this common sexually transmitted disease.
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Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat
More LessThe nucleotide sequence of 42090 bp of vaccinia virus strain WR is presented. The sequence includes the SalI L, F, G and I fragments and starts near the centre of the HindIII A fragment and extends rightwards towards the genomic terminus, finishing approximately 0.5 kb internal of the inverted terminal repeat (ITR). Translation of this region has identified 65 open reading frames (ORFs) of greater than 65 amino acids in length. Fifty-one of these which do not extensively overlap other larger ORFs have been subjected to further analysis; the other 14 are termed minor ORFs. In the rightmost 28.7 kb, the genes are, with one exception, transcribed towards the genomic terminus, similar to the arrangement of genes at the left end of the virus genome. Internal of this region the genes are expressed off either DNA strand but still predominately rightwards. ORFs are tightly packed with few intergenic non-coding regions of greater than 250 bp. Protein sequence comparisons have established a remarkably high number of homologies with entries in existing protein databases. Of these, DNA ligase, thymidylate kinase, two serine-threonine protein kinases, two serine proteinase inhibitors (serpins), two interleukin-1 receptor homologues and a discontinuous ORF related to tumour necrosis factor receptor have been reported. Other homologies include lectins, profilin, 3β-hydroxy steroid dehydrogenase, superoxide dismutase, guanylate kinase, ankyrin and complement factor H. In addition, there are a number of polypeptides with predicted properties of membrane-associated, secretory or glyco-proteins. Twelve gene families are described here and elsewhere. There is considerable similarity between genes from the right and left end of the virus genome that may have arisen by terminal transposition events. Several differences from the corresponding region of vaccinia virus strain Copenhagen sequence are noted. Near the right terminus the sequences diverge completely, and internal of this there are multiple examples of deletion of short sequences (eight to 10 nucleotides) that lie within penta- or hexanucleotide direct repeats.
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Differentiation of herpes simplex virus-induced fusion from without and fusion from within by cyclosporin A and compound 48/80
More LessTreating strains of herpes simplex virus (HSV) in culture with either cyclosporin A or compound 48/80, allowed the strains to be divided into two groups. Group 1 contains the strains ANG and HFEM of HSV-1 and Lux syn (HSV-2) producing fusion from within (FFWI) and fusion from without (FFWO). Cyclosporin A fails to inhibit both types of fusion at concentrations up to 100 µm. Strains ANG and HFEM belong to the syn 3 marker locus group identified for HSV-1. Group 2 contains all other fusion-producing strains of HSV tested so far. Cyclosporin A inhibits FFWI at concentrations as low as 10 to 20 µm. These strains belong to the syn locus marker groups 1, 2, 4 and 5. From the fact that mutations in glycoprotein B belong to the syn 3 marker group we conclude that glycoprotein B is of major importance for FFWO. Compound 48/80 also differentiates between these two groups of viruses. O-Acetyl cyclosporin A is unable to inhibit FFWI induced by group 2 viruses; in contrast, cyclosporin H and the Ca2+ ionophore A23187 exert inhibition effects similar to those exerted by cyclosporin A. We conclude from the effects of these compounds that binding properties of the OH group of cyclosporin A and an increase of Ca2+ ions may be preconditions for the observed effects. Binding of cyclosporin A to cyclophilin does not appear to be responsible for these effects.
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Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli
The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3.5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5.3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0.2 to 0.5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR.
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Development of immunogenic recombinant Oka varicella vaccine expressing hepatitis B virus surface antigen
Recombinant Oka varicella vaccine expressing hepatitis B virus (HBV) surface antigen (HBs) was constructed by inserting the HBs gene into the viral thymidine kinase (TK) gene and was examined for its immunogenicity in guinea-pigs. The HBs gene encoding 25 amino acids of preS2 and the whole of the S region was inserted into the TK gene of the cloned plasmid. The chimeric plasmid DNA and Oka varicella vaccine DNA were cotransfected and recombinant virus was isolated after immunofluorescence screening using a monoclonal antibody to HBs and a fluorescein-conjugated anti-mouse antibody. Expression of viral HBs was detected in the cytoplasm of infected cells and was stable over several repeated passages in vitro. The recombinant virus expressed 26K and 30K HBs molecules in infected cells and the culture supernatant contained 30K and 35K HBs molecules. HBs was purified at a density of 1.20 g/ml from the culture supernatants. The recombinant virus induced an antibody response to HBs as well as to varicella-zoster virus (VZV) in guinea-pigs, and the antibody titre to HBs was comparable to that induced by a recombinant HBs subunit vaccine produced in yeast. Thus a single dose of live recombinant Oka varicella vaccine could induce good immunity to VZV and HBs. The recombinant Oka varicella vaccine expressing HBs may be a good candidate for a combined HBV and VZV vaccine.
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Latent human herpesvirus 6 infection of human monocytes/macrophages
More LessHuman herpesvirus 6 (HHV-6) DNA was detected in peripheral blood from exanthem subitum patients during the acute and convalescent phases of infection using the polymerase chain reaction. Although DNA could be detected in non-adherent and adherent mononuclear cells during the acute phase, it was detected predominantly in adherent cells during the convalescent phase; furthermore, viral DNA was found in adherent cells of healthy adults. When adherent mononuclear cells were cultured in vitro, virus was found to replicate well in differentiated cells cultured for 7 days in vitro before infection. When cells were cultured for more than 1 month, no detectable antigen and no evidence of virus growth was observed, but viral DNA could be detected. These apparently latently infected monocytes were treated with phorbol ester, after which virus could be recovered from the cultures. Therefore, we have developed an in vitro latency system for HHV-6; our results suggest that HHV-6 may latently infect monocytes in vivo and in vitro and that it may be reactivated in cells by some factors.
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Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides
More LessThe entire amino acid sequence of human cytomegalovirus (HCMV) 150K matrix phosphoprotein (pp150), consisting of 1048 amino acid residues, was divided into 95 overlapping 20 amino acid peptides which were synthesized on polyethylene rods. The rods were subjected to ELISA with pooled anti-HCMV-positive and anti-HCMV-negative sera. Four peptides recognized by the anti-HCMV-positive pool only were synthesized by the solid-phase method and their reactivity in a conventional ELISA, using a panel of 14 individual anti-HCMV-negative and 20 anti-HCMV-positive antisera, was evaluated; three peptides were found to be specifically reactive. Results obtained with one of these peptides (residues 595 to 614) in ELISA showed a good correlation with those obtained using a routinely performed complement fixation test.
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Subcellular distribution of the major immediate early proteins of human cytomegalovirus changes during infection
More LessThe 72K immediate early (IE) 1 protein of human cytomegalovirus and the 68K protein, also encoded by the IE1 gene, were detected by immunoelectron microscopy using monoclonal antibodies specific for the 68K or 72K proteins. Early after infection, both proteins were localized to the nucleus, but with different localization patterns. Late after infection, both of the proteins decreased markedly in the nucleus, in which nucleocapsids appeared. Simultaneously, the 68K protein became diffusely distributed in the cytoplasm and the plasma membrane, whereas the 72K protein was distributed in the nuclear envelope and the plasma membrane. Both proteins were also observed in the coating membrane of the extracellular dense bodies.
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Characterization of a 52K protein of murine cytomegalovirus and its immunological cross-reactivity with the DNA-binding protein ICP36 of human cytomegalovirus
We have developed a hybridoma, designated 25G11, which produced a monoclonal antibody (MAb) reactive with a 52K protein of murine cytomegalovirus (MCMV). This MAb, 25G11, was reactive with a protein band of 52K in MCMV-infected cell lysates and with a protein of 49K in human CMV (HCMV)-infected cell lysates as detected by immunoblot analysis. With purified MCMV virions, 25G11 gave a faintly immunoreactive band of 52K. However, no immunoreactive protein band was detected with purified HCMV virions, nor with purified HCMV or MCMV envelope preparations. By immunocytochemistry, 25G11 detected viral antigen primarily in the nucleus of HCMV- or MCMV-infected cells. The antibody 25G11 was used to screen a λgt11 library of HCMV DNA fragments. One of the isolated clones (λ32323B) was employed for gene mapping on the HCMV genome, which suggested that the immunoreactive HCMV protein was the DNA-binding protein (ICP36). Analysis of the recombinant fusion protein with antibody 25G11 and with an MAb (CH16) specific for an HCMV DNA-binding protein confirmed the identity of the cross-reacting protein as ICP36. Furthermore, we found that whereas the epitope recognized by 25G11 was conserved between HCMV and MCMV proteins, the epitope recognized by CH16 was unique to HCMV and thus represents a variable region in the protein.
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Temporal control of bovine herpesvirus type 4 glycoprotein synthesis
More LessThe glycoprotein gp6/gp10/gp17, gp11/VP24 and gp8 are major bovine herpesvirus type 4 antigens. The temporal expression of these glycoproteins was studied and it was shown that gp6/gp10/gp17 appeared as early as 6 h post-inoculation (p.i.), and gp11/VP24 and gp8 were detected 8 h p.i. Moreover, a precursor of two components of gp6/gp10/gp17 glycoprotein [p(gp10/gp17)] was a beta-gamma protein, whereas the gp11/VP24 and gp8 glycoproteins were gamma proteins.
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Loss of pseudorabies virus thymidine kinase activity due to a single base mutation and amino acid substitution
More LessThe nucleotide sequences of the coding region of the thymidine kinase (TK) gene of pseudorabies virus strain NIA3 and a TK− mutant (ATK5) were determined. The coding region of the TK gene consists of 320 codons capable of producing a polypeptide with an M r of 34979. The mutant expressed an inactive TK polypeptide when translated in vitro; a unique base substitution was detected in the mutant TK modifying amino acid position 13, at which aspartic acid replaces glycine. This modification affects the nucleotide-binding site, thus explaining the expression of an inactive TK polypeptide.
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Role of viral ribonucleotide reductase in the increase of dTTP pool size in herpes simplex virus-infected Vero cells
More LessInfection of Vero cells with herpes simplex virus (HSV) causes a marked increase in the dTTP pool size of infected cells. In this study we examined the relative importance of the HSV-encoded ribonucleotide reductase (RR) and thymidine kinase (TK) in the increase of dTTP. In cells infected with an RR deletion mutant of HSV-1 strain KOS, there was no significant increase in the size of the dTTP pool, whereas the dTTP pool in HSV-1(TK−)-infected cells was increased in size to almost the same extent as that in HSV-1(TK+)-infected cells. Moreover, it was found that the increase in dTTP pool size was strongly inhibited by the addition of hydroxyurea, a specific inhibitor of RR, and 5-fluoro-2′-deoxyuridine, a specific inhibitor of thymidylate synthetase. These results suggest that the induction of viral RR is of primary importance in the increase of dTTP pool size in HSV-1-infected Vero cells.
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Immunogenicity in mice of varicella-zoster virus glycoprotein I expressed by a vaccinia virus-varicella-zoster virus recombinant
More LessSeveral vaccinia virus (VV)-varicella-zoster virus (VZV) recombinants expressing glycoprotein I (gpI) of VZV were isolated from the Prague strain of VV. One of these, v46, was inoculated intraperitoneally into mice. Groups of mice were bled 4 and 8 weeks later and their sera were examined for anti-VZV and anti-VV antibodies by ELISA. At 4 weeks, all mice inoculated with the three largest virus doses (107, 106 and 105 p.f.u.), and at 8 weeks all mice inoculated with the four highest virus doses (107, 106, 105 and 104 p.f.u.), had developed both anti-VV and anti-VZV antibodies. Antibodies were also detected in a high proportion of mice infected with lower doses of virus and in some instances VZV antibodies were present in the absence of VV antibodies. None of the animals inoculated in parallel with either a thymidine kinase-negative mutant of the original VV or diluent alone developed antibody reactive with VZV. The specificity of the reaction was assessed further by Western blotting using anti-gpI monoclonal antibodies as a positive control. Sera from animals immunized with v46 possessed antibody capable of neutralizing extracellular VZV in the presence of complement.
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17D yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein
We have constructed a recombinant baculovirus containing cloned DNA encoding the membrane and envelope (E) proteins of 17D yellow fever vaccine virus. Spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66K protein which corresponded to the estimated size of the protein encoded by the cloned inserted DNA, and a 54K protein with the same molecular size as that of the authentic 17D yellow fever virus E protein. This recombinant 54K protein was labile, producing E protein-specific breakdown products (45K to 36K). Indirect immunofluorescence, using a panel of E protein-specific monoclonal antibodies, showed that the recombinant protein was presented both inside as well as on the surface of cells and was antigenically indistinguishable from the E protein of 17D yellow fever vaccine virus.
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Location of phosphorylated residues in human respiratory syncytial virus phosphoprotein
More LessThe phosphoprotein (P protein) from human respiratory syncytial virus Long strain, labelled in vivo with [32P]orthophosphate, was purified from virions or virus-infected human epithelial (Hep-2) cells. The main phosphorylated amino acid found was serine. The determination of the N-terminal sequence of unphosphorylated and phosphorylated fragments of P protein obtained after chemical or enzymic treatments suggested that some or all of the six serines present at positions 116, 117, 119, 143, 156 and 161 are the major phosphorylated residues, although a modification in serine residues at positions 86, 94 and 99 can not be ruled out.
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The M2 protein of influenza A virus is acylated
M. Veit, H.-D. Klenk, A. Kendal and R. RottThe M2 protein of influenza A virus, a 97 amino acid integral membrane protein expressed on the surface of infected cells, is covalently modified with long chain fatty acids. The fatty acid bond is sensitive to treatment with neutral hydroxylamine and mercaptoethanol, which indicates a labile thioester type linkage. Thinlayer chromatographic fatty acid analysis of [3H]myristic and [3H]palmitic acid-labelled M2 protein shows that palmitic acid is the predominant fatty acid linked to this polypeptide. Palmitoylation of M2 occurs post-translationally and causes an upward shift in the SDS-PAGE mobility of the protein.
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Individual adenovirus E1B proteins induce transformation independently but by additive pathways
More LessThe specific contributions of human adenovirus type 5 early region 1B (E1B) proteins were examined using mutants which synthesize these products individually. In cooperation with E1A, transformation of primary baby rat kidney cells was achieved with either the 176R protein or 496R protein alone, albeit at an efficiency considerably less than that observed when both were present. These results indicate that transformation mediated by either E1B product can proceed independently, but that the processes involved are additive.
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Identification of a new viroid as the putative causal agent of pear blister canker disease
More LessA small RNA species with the structural and functional properties characteristic of viroids has been isolated from three different pear sources each of which induced symptoms of the pear blister canker (PBC) disease when indexed in the pear indicator A 20. A close association between this RNA and PBC disease was established, since two of the three studied sources were known to be affected by this malady only, and the viroid was not detected in healthy pear tissue. Moreover, the PBC-associated viroid (PBCVd) replicated when purified preparations were inoculated into cucumber and pear plants. PBCVd behaved in denaturing polyacrylamide gels as a circular RNA with a molecular size of approximately 315 nucleotide residues. Analysis by dot blot hybridization indicated that PBCVd shares similarities in sequence with peach latent mosaic viroid and hop stunt viroid, and to a lesser extent with apple scar skin viroid.
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Mutagenesis of the AC3 open reading frame of African cassava mosaic virus DNA A reduces DNA B replication and ameliorates disease symptoms
More LessSmall insertions were made independently at each of four unique restriction sites on African cassava mosaic virus (ACMV) DNA A to disrupt the three overlapping complementary-sense open reading frames (ORFs) herein designated AC1, AC2 and AC3. The DNA A mutants were assayed for their infectivity by agroinoculation of monomeric constructs to Nicotiana benthamiana plants containing chromosomal insertions of ACMV DNA B. Disruption of the AC3 ORF alone resulted in a delay and amelioration of disease symptoms which correlated with reduced replication of DNA B. Normal replication of DNA A still carrying the AC3 ORF mutation was found in extracts from these plants. No ACMV DNA or symptoms were observed in corresponding inoculations with either the simultaneous disruption of the overlapping AC2 and AC3 ORFs or disruption of the AC1 ORF. Complementation by the inoculation of different mutant pairs produced a delay in disease symptoms followed by repair of mutated sites. A DNA A construct with the virus-sense AV1 (coat protein) ORF deleted was infectious producing typical ACMV disease symptoms. A similar construct with a larger deletion encompassing the complementary-sense AC3 ORF produced symptomless infections. The DNA recovered from plants revealed DNA A of normal size where the position of the deleted ORF was replaced with cloning vector DNA. Significantly reduced DNA B replication was observed for the AC3 deletion construct.
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Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium-mediated transfer
In nature, rice tungro disease is caused by an RNA and a DNA virus complex, but we have obtained an independently infectious clone of rice tungro bacilliform virus (RTBV) DNA. Infectivity could be demonstrated only when a more than unit-length copy was cloned in the Agrobacterium binary vector Bin19 and agroinoculated into rice plants. Rice plants thus agroinfected with cloned RTBV DNA showed typical symptoms of tungro disease, presence of viral DNA and bacilliform particles, and could be used as a source of virus to infect healthy plants by the green leafhopper (Nephotettix virescens). The importance of this infectious clone in understanding the molecular biology of RTBV and the rice tungro disease is discussed.
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In vivo expression of the 29000 M r protein from RNA-2 of pea early browning tobravirus
More LessThe purpose of this work has been to investigate in vivo transcription and translation products from an open reading frame (ORF) located downstream of the coat protein (CP) cistron on RNA-2 of pea early browning virus (PEBV). This work was initiated as a step towards elucidation of the significance of this putative gene. Sequence data on RNA-2 suggest that a 29600 M r (29.6K) protein is translated from the ORF in question. Hybridization with ORF-specific probes on Northern blots with RNA from infected plants showed that PEBV synthesizes a subgenomic RNA encoding CP (RNA-2a) with a size of 3000 nucleotides (nt) and that a putative subgenomic RNA (RNA-2b) encoding the 29.6K protein appears to have a size of 1600 nt. This is 300 nt less than the size predicted from the sequencing data. For antibody production, a cDNA fragment harbouring 85% of the 29.6K ORF was cloned into the pUEX3 expression vector. The resulting plasmid expresses the 29.6K protein as a fusion protein with β-galactosidase and this protein was used for raising antiserum containing specific anti-29.6K protein antibodies. By using these antibodies on immunoblots it was demonstrated that the 29.6K protein is expressed in infected plants.
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Use of group-specific primers and the polymerase chain reaction for the detection and identification of luteoviruses
More LessA general diagnostic assay for a number of distinct luteoviruses was developed using the polymerase chain reaction (PCR) and restriction enzyme analysis. Two minimally degenerate, group-specific primers were derived from previously published RNA sequences of three luteoviruses. This primer pair generated specific PCR fragments of about 530 bp from extracts of plants infected with potato leafroll virus, beet western yellows virus, or New York barley yellow dwarf virus (BYDV) serotypes MAV, PAV, RMV, RPV and SGV, which span much of the respective viral coat protein gene. Each virus was easily distinguished from the others by restriction enzyme analysis of the amplified DNA products. Samples from BYDV-infected oat and wheat collected in Nebraska were identified as containing PAV-like serotypes; micro-heterogeneity was detected in several samples. This method provides a rapid, sensitive and relatively inexpensive means of luteovirus detection and identification. It is the first test capable of simultaneously detecting all five BYDV serotypes.
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Secondary structures of narcissus mosaic virus coat protein
Circular dichroism (CD) measurements on the coat protein of narcissus mosaic virus particles show that the dominant secondary structure is the α-helix, with a 45 (±2)% content. The β-sheet content is much lower at 5 (±3)%. Both values are essentially the same as those found in potato virus X coat protein. The CD results are used to assess the results of secondary structure prediction methods.
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