- Volume 74, Issue 7, 1993
Volume 74, Issue 7, 1993
- Animal
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Induction of cytolytic T lymphocytes directed towards the V3 loop of the human immunodeficiency virus type 1 external glycoprotein gp120 by p55gag/V3 chimeric vaccinia viruses
More LessT cell-mediated cytotoxicity may play an important role in controlling infection by human immunodeficiency virus (HIV). In order to study the ability of rationally designed antigens to induce cytolytic T lymphocytes (CTLs) we replaced stretches of 30 to 50 amino acids at the p17-MA/p24-CA cleavage site, within the p24-CA moiety and within the p6-LI portion of the HIV type 1 p55 gag precursor by the third variable domain (V3) of the external glycoprotein gp120. This site is known to be a target for CTL attack in mice and humans. The chimeric antigens were recombined into highly attenuated vaccinia viruses in order to investigate class I major histocompatibility complex (MHC)-restricted presentation of antigenic V3 peptides. Immunoprecipitation and Western blot analysis of the group-specific antigen (p55 gag )/V3 chimeric proteins demonstrated significant differences in the accessibility of the V3 domain for a monoclonal antibody or polyclonal V3-specific antisera, depending on the position of the V3 loop within the p55 gag carrier protein. Immunization of BALB/c mice with three variants of p55 gag /V3 recombinant vaccinia virus, however, resulted in a comparable priming of CD4− CD8+ CTLs in vivo irrelevant of the position of the V3 loop within p55 gag . Local conformational changes, including the V3 domain within the p55 gag /V3 chimeras, did not demonstrate a significant effect on V3-specific lysis of the target cells when compared to the authentic gp120 envelope protein. Class I MHC-restricted CTLs induced by a V3 consensus sequence cross-reacted perfectly with the LAI strain-derived V3 loop sequence. These data indicate that the combination of selected epitopes (V3) with immunologically relevant complex carrier proteins (p55 gag ) can be accomplished without the loss of biological activity.
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Tumour necrosis factor-α increases the sensitivity of human immunodeficiency virus (HIV)-infected monocytic U937 cells to the complement-dependent cytotoxicity of sera from HIV type 1-infected individuals; role of the gp120 protein
More LessSera of 40 intravenous drug addicts [25 seropositive and 15 seronegative for human immunodeficiency virus (HIV)] were tested for the presence of cytotoxic antibodies against uninfected and HIV-infected monocytic U937 cells. Six of the 25 seropositive samples proved to be cytotoxic for HIV-infected target cells in the presence of complement. The pretreatment of HIV-infected U937 cells with tumour necrosis factor (TNF)-α (which enhances virus production in these cells) increased the detection of serum cytotoxicity and 60% of these sera became cytotoxic. The percentage lysis was also increased after the TNF-α treatment of the target cells (from 16·2 ± 4·5 to 71·2 ± 4·9). The complement-dependent cytotoxic activity of these sera was significantly reduced by pretreatment with recombinant HIV gp120 antigen. This reduction was dose-dependent in the majority of cases. Immunofluorescence studies suggested that the cytotoxic sera mainly interacted with the viral antigens localized on the membrane of HIV-infected TNF-treated U937 cells. Moreover, comparative Western blot analyses using cellular extracts from untreated and TNF-treated U937 cells showed that there was a positive correlation between the cytotoxic phenotype and the capacity of sera to recognize the gp120 protein in extracts from TNF-treated HIV-infected cells. These results suggest that in some circumstances endogenous TNF-α can be a protective factor because it can render persistently infected cells highly sensitive to complement-dependent serum cytotoxicity as a result of increased expression of the relevant viral antigen (gp120) on the cell membrane.
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CD4-independent infection of human peripheral blood dendritic cells with isolates of human immunodeficiency virus type 1
Dendritic cells (DC) are members of a distinct family of bone marrow-derived leukocytes. DC are potent accessory cells for a number of T cell-mediated immune responses, including autologous and allogeneic mixed leukocyte reactions, and mitogen- and antigen-stimulated lymphocyte proliferation. In the present study, DC purified from human peripheral blood were inoculated with various strains (IIIB, SF2, WMJ1, SF162, 89∙6 and clone HXB2) of human immunodeficiency virus type 1 (HIV-1) displaying different patterns of cellular tropism. Viral replication was demonstrated by detection of p24 antigen (Ag) intracellularly and in culture supernatants, and by Southern and Northern blot analyses for the presence of HIV DNA and RNA, respectively, within infected cells. Cell-free and cell-associated p24 Ag levels rose substantially when DC were inoculated with strains SF162, 89∙6 and clone HXB2. In contrast, p24 Ag levels rose only marginally after inoculation of DC with strains IIIB, SF2 and WMJ1. Purified DC did not express detectable membrane CD4, although CD4 mRNA was detected by reverse transcriptase PCR. The presence of anti-CD4 monoclonal antibodies failed to block infection of DC by any of the HIV strains tested, suggesting the existence of a CD4-independent alternative pathway of viral entry. The possibility that DC serve as a reservoir for HIV-1 must be considered.
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An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis
More LessThe murine coronavirus neurotropic strain JHM (MHV-JHM) nucleocapsid (N) protein induces a strong T-helper cell response in Lewis rats. It has been shown previously that N-specific CD4+ T cells can confer protection against acute disease upon transfer to otherwise lethally infected rats. To define the major antigenic regions that elicit this T cell response, truncated fragments of N protein were expressed from a bacterial expression vector and employed as T cell antigens. Lymphocytes from either MHV-JHM-infected or immunized rats were stimulated in culture with virus antigen, grown and tested for their specificity to the N protein fragments. The carboxy-terminally located C4-N fragment (95 amino acids) induced the most pronounced proliferative response irrespective of whether the lymphocyte culture was derived from immunized or MHV-JHM-infected rats. We established T cell lines specific for the truncated N protein fragments and tested their potential to mediate protection by transfer experiments. Only the T cell line C4-N and the T cell line specific for the full-length N protein were protective. By contrast, all truncated N protein fragments elicited a humoral immune response and contained antigenic sites recognized by antibodies from diseased rats.
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Characterization of monoclonal antibodies raised against a synthetic peptide capable of inducing a neutralizing response to human rhinovirus type 2
More LessSynthetic peptides incorporating the derived amino acid sequence of VP2 residues 156 to 170 of human rhinovirus type 2 (HRV2) have previously been shown to elicit antibodies that neutralize virus infectivity. The proportion of virus-reactive antibodies present in polyclonal antisera to these peptides is, however, very low. Moreover, neutralization titrations of such antisera correlate poorly with other assays of either anti-virus or anti-peptide activity, suggesting the presence of antibodies with different specificities. To investigate these findings further, we produced a panel of monoclonal antibodies (MAbs) to VP2 peptides of residues 156 to 170 and characterized their reactions with a range of antigens in ELISA, precipitation and neutralization titrations. All the MAbs obtained recognized the homologous peptide, but could be divided into four main reaction groups according to their specificity for viral antigens. Antibodies in the first group recognized and neutralized native virus, apparently by preventing attachment to cells. A second group of MAbs bound to intact particles with similar affinities to the first group, but failed to neutralize infectivity. Antibodies in the third group recognized virus only after capsid distortions incurred by heating or by previous reaction with polyclonal antibodies. The fourth group comprised MAbs that were mainly peptide-specific. Some possible applications of anti-peptide MAbs to improving the design of peptide immunogens are considered.
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Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidemica: a novel method using antigen-coated magnetic beads for specific B cell isolation
More LessHuman monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein—Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four antiglycoprotein 2-specific clones. Identical isotypes (IgGl λ) and isoelectric points (9·2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human × mouse partner SPAM-8. Several of the established human × (human × mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.
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Influenza A virus haemagglutinin polymorphism: pleiotropic antigenic variants of A/Shanghai/11/87 (H3N2) virus selected as high yield reassortants
More LessGenetic reassortment of the A/Shanghai/11/87 (H3N2) variant of influenza A virus with A/PR8/34 (H1N1) virus [the standard donor of high yield (hy) genes for influenza vaccine viruses] resulted in the isolation of two reassortants with differing H3 haemagglutinin (HA) phenotypes, X-99 and X-99a. The two HA phenotypes were derived from individual subpopulations of the H3N2 wild-type virus during the reassortment event. The HA mutants and their respectively derived reassortants (identical in RNA genotype) differed in antigenicity, replication characteristics, yield in chick embryos and haemagglutinin gene sequence. Despite antigenic differences in reactions to polyclonal rabbit antisera of 60%, both X-99 and X-99a, the hy reassortants, were equally immunogenic and protective in BALB/c mice to challenge by parental wild-type virus. Differences in HA phenotype were related to a Ser to Ile change at amino acid position 186. These findings emphasize the polymorphism of influenza virus strains as well as the need for caution in selection of vaccine strains from among antigenically distinct viral subpopulations.
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Splicing of influenza virus matrix protein mRNA expressed from a simian virus 40 recombinant
More LessInfluenza virus RNA segment 7 encodes two proteins, M1 and M2, depending on the optional removal of an intron from its primary transcript. To investigate the mechanism of this regulated splicing, an influenza virus segment 7 cDNA was cloned under the control of simian 40 virus (SV40) early promoter and poly(A) signals in an SV40 recombinant virus (SVM), and expressed in COS-1 cells. Expression of both M1 and M2 proteins was detected in SVM-infected cells, suggesting (i) the appropriate splicing events to generate M2 mRNA occur in these cells and (ii) significant amounts of unspliced M1 mRNA are transported to the cytoplasm. Analysis of the relative proportion of M2 mRNA to mRNA3 indicated that the use of the alternative 5′ splice sites is reversed in SVM-infected cells compared with those infected with influenza virus. In addition, a different intranuclear distribution of segment 7 transcripts was found in each type of infected cell. We speculate that these differences in splicing efficiency and splice site choice might be related to different subnuclear localizations of segment 7 transcripts synthesized by the different transcriptional machineries.
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Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter
More LessA previous study of the 12 nucleotide-long influenza A virion RNA promoter has shown that three nucleotides, residues 9 to 11, were crucial for transcription in vitro, although other nucleotides play a significant but less important role. A model for polymerase-promoter recognition was proposed, according to which there were two sites: a binding site at residues 9 to 11 and a regulatory site at or near the site of initiation at residue 1. By studying the effect of point mutations in the promoter on the binding efficiency of the polymerase using a photochemical cross-linking assay, we now show that residues 9 to 12 are crucial for binding. In addition residues 4 to 8, though not as important, are involved in binding, possibly by stabilizing the polymerase-promoter complex. Both PB1 and PB2 apparently play an important role during virion RNA promoter recognition and binding.
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Immunochemical structure of the carboxy-terminal part of hepatitis B e antigen: identification of internal and surface-exposed sequences
The C-terminal region of hepatitis B virus (HBV) e antigen (HBeAg), amino acids (aa) 121 to 147, was characterized for reactivity with 15 monoclonal antibodies (MAbs) and sera from 16 chronic carriers on the HB surface antigen with anti-HBe. Recombinant proteins exposing fragments of the HBc/e polypeptide (aa 29 to 176, 60 to 176, 101 to 176, 121 to 176, 134 to 176, 138 to 176, 139 to 176, 140 to 176, 146 to 176 and 156 to 176) fused to the N terminus of the coat protein of RNA phage fr were constructed, as were two sets of synthetic peptides covering residues 121 to 136 and 130 to 147, where each residue was sequentially substituted by alanine. The MAbs were found to recognize overlapping epitopes in the fusion proteins within residues 121 to 176; however, none of the MAbs reacted with proteins covering residues 146 to 176 and 156 to 176. Using the synthetic peptides it was found that the MAbs recognized epitopes at residues 128-TPPAYR-133, 133-RPPNAP-138, 135-PNAPIL-140, 138-PILSTLPE-145 and 143-LPET-146. Only MAbs recognizing the epitope 128-TPPAYR-133 were found to react with both native HBeAg and denatured HBcAG, whereas MAbs recognizing epitopes located closer to the C terminus of HBeAg were reactive only with denatured HBcAg. The recognition sites for the human IgG1 overlapped with the epitopes of the MAbs recognizing native HBeAg. Our interpretation of these findings is that the region 124 to 133 is on the surface of native HBeAg and denatured HBcAg, and that the adjacent region 135 to 147 is not accessible on the surface of native HBeAg, but becomes exposed on denatured HBcAg.
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Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen
A 681 nucleotide fragment of the hepatitis B virus (HBV) genome was sequenced that corresponded to the complete gene for hepatitis B surface antigen (HBsAg) in 80 HBsAg- and hepatitis B e antigen (HBeAg)-positive sera of diverse geographical origins. These and 42 previously published HBV sequences within the S gene were used for the construction of a dendrogram. In this comparison, each of the 122 HBsAg genes was found to be related to one or other of the six previously identified genomic groups of HBV, A to F. The HBV strains within each genomic group showed a characteristic geographical distribution. Group A genomes were represented by 23 strains mainly originating in northern Europe and sub-Saharan Africa. The group B and C genomes, represented by 17 and 28 strains respectively, were confined to populations with origins in eastern Asia and the Far East. The group D genomes, represented by 38 strains, were found worldwide, but were the predominant strains in the Mediterranean area, the Near and Middle East, and in south Asia. Group E genomes, represented by nine strains, were indigenous to western sub-Saharan Africa as far south as Angola. There were indications that the F group, made up of six strains, represented the genomic group of HBV among populations with origins in the New World. Thus, HBV has diverged into genomic groups according to the distribution of mankind in the different continents. As well as giving information on the genetic relationship of HBV strains of different geographical origin, this study also provides information on the primary structure of HBsAg in different regions of the world. Such data might prove valuable in explaining the reported failures to obtain protection with current HBV vaccines.
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A mutational analysis of the DNA-binding domain of the herpes simplex virus type 1 UL9 protein
More LessThe herpes simplex virus type 1 origin-binding protein is encoded by gene UL9. We previously described a plasmid, pB1, which encodes a fusion protein containing only the C-terminal 317 amino acids of the UL9 polypeptide and showed that this product retains sequence-specific DNA-binding ability. Two series of pB1 mutants have now been constructed and the polypeptides were tested for origin-binding activity. Using C-terminal truncations, we show that the C-terminal 34 amino acids of UL9 are dispensable for binding and that essential residues lie between positions 801 and 818. Analysis of a series of mutants containing insertions of four amino acids at various positions identified regions of the DNA-binding domain in which alterations either abolished or had relatively little effect upon binding activity. Two mutants which were intermediate in their binding activities also exhibited temperature- or sequence-specific effects.
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Protection against ocular and cutaneous infection with herpes simplex virus type 1 by intragastric immunization with live virus
More LessIntragastric administration of live herpes simplex virus type 1 (HSV-1) was assessed for the induction of humoral immune responses and for protection against ocular and cutaneous challenge with virus. Mice showed no clinical abnormalities following intragastric inoculation with three different strains of virus (Miyama + GC, SC16, and P2C6, a thymidine kinase-defective mutant). Replication of virus was not detected in the oesophagus, superior cervical ganglia or coeliac ganglia of such animals and latent infection was not detected in these ganglia at later times after inoculation. Induction of a mucosal immune response was indicated by the presence of antibody (mainly IgG or IgA)-secreting cells in Peyerʹs patches. Intragastric immunization gave protection to some extent against ocular challenge and to a greater extent against cutaneous challenge with HSV-1. Following the latter challenge, particularly after intragastric immunization with strains SC16 and Miyama, the establishment of latency was almost completely prevented.
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Intranuclear foci containing low abundance herpes simplex virus latency-associated transcripts visualized by non-isotopic in situ hybridization
More LessDuring latent infection of neurons with herpes simplex virus type 1 (HSV-1), several RNA transcripts of varying abundance arise from a single locus within the virus repeats. The functions of latency-associated transcripts (LATs) are unknown and the relationship between the various RNA species requires further clarification. Reported here is a novel approach to the study of HSV transcripts during latency, based on the increasing realization that cellular and viral RNAs are synthesized and processed by macromolecular complexes that occupy discrete compartments within the nucleoplasm of a cell. High resolution non-isotopic in situ hybridization was used to study the intranuclear topology of HSV-1 LATs in primary sensory neurons of latently infected mice and humans. Low abundance (minor) LATs were localized to sharply defined intranuclear foci of 1 to 3 μm in diameter. On average, there were 2·6 to 2·8 foci/LAT+ neuronal profile (5 μm), representing 13 to 14 foci/cell. In contrast to the focal deployment of minor LATs, the more abundant latency-associated RNAs were distributed diffusely throughout the nucleoplasms of latency infected neurons, with prominent sparing of nucleolar regions. These data establish a foundation for studying the synthesis, processing and transport of LATs in vivo. It should now be possible to investigate the nature of those cellular products which associate with HSV-1 encoded LATs in vivo and thereby determine whether minor LATs are associated with previously characterized macromolecular complexes, such as those responsible for processing of pre-messenger RNA.
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Stable constitutive expression of glycoprotein B (gpUL55) of human cytomegalovirus in permissive astrocytoma cells
More LessPermanent cell lines showing homogeneous constitutive expression of glycoprotein B (gpUL55; gB) of human cytomegalovirus (HCMV) were selected, in the presence of geneticin, from human astrocytoma cells (U373) after transfection with recombinant pRC/CMV-gB carrying the complete coding sequence for HCMV gB and for aminoglycoside phosphotransferase. The biosynthesis and processing including specific proteolytic cleavage, formation of disulphide-linked oligomers as well as transport of recombinant gB in three of four established transformed cell lines essentially resembled that found in infected parental U373 except for eventual degradation after 2 h of gB synthesis. Analysis of the fourth transformant expressing uncleaved gB suggested that proteolytic cleavage is not required for normal intracellular transport. The stable transformants retained permissiveness for productive superinfection with HCMV. The application of cell lines transformed with mutagenized HCMV gB for the rescue of genetically engineered HCMV mutants is discussed.
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Dilated heart failure in transgenic mice expressing the Epstein—Barr virus nuclear antigen-leader protein
More LessThe Epstein—Barr virus nuclear antigen-leader protein (EBNA-LP) is required for high efficiency B lymphocyte growth transformation by the virus. To test the potential contribution of EBNA-LP to tumorigenesis in vivo, we produced transgenic mice carrying an EBNA-LP cDNA construct, using the widely expressed metallothionein promoter. Expression of EBNA-LP was detected in liver, kidney, heart, lung and spleen. There were no apparent oncogenic consequences of EBNA-LP expression. Unexpectedly however, at ages ranging from about 4 months to over a year, transgenic mice developed symptoms of congestive heart failure, including left ventricular dilatation, right ventricular hypertrophy, left atrial thrombosis, pulmonary oedema and hydrothorax. Fibrillation was not apparent in the electrocardiograph; however a reduction in T-wave amplitude suggested that the development of an abnormality of ventricular repolarization may precede the manifestation of overt symptoms. The highly predictable development of dilated heart failure in these transgenic mice suggests they may be a useful model for the pathophysiological changes associated with human dilated cardiomyopathy.
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Integration of a short Epstein—Barr virus DNA fragment in a B95-8 virus converted Burkitt lymphoma line expressing Epstein—Barr nuclear antigens EBNA2 and EBNA5
We have analysed the expression of transformation-associated viral antigens, the Epstein–Barr virus (EBV) DNA content and the phenotypic characteristics of two B95-8 virus-converted sublines of the EBV-negative Burkittʹs lymphoma (BL) line BL28. The converted lines called E95A-BL28 and E95B-BL28, respectively, differed in their EBV gene expression. The E95B convertant expressed virus-encoded nuclear antigens EBNA1 to -6 and the membrane protein LMP1, but only EBNA2 and EBNA5 were detected by immunofluorescence and immunoblotting in the E95A convertant. Only the entire BamHI W, Y and H regions could be detected in the E95A convertant by hybridization of Southern blots with probes covering the BamHI C, W, Y, H, F, E, K and Nhet regions of the EBV genome. EBV episomes were found to be absent in the E95A convertant as seen by Gardella gels. The E95A convertant retained the phenotypic characteristics of the EBV-negative parental line, and remained highly clonable in agarose. In contrast, expression of EBNA1 to -6 and LMP1 was accompanied by a shift towards a more lymphoblastoid cell line-like phenotype and by loss of agarose clonability in the E95B convertant.
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Protective activity of the lipid A analogue GLA-60 against murine cytomegalovirus infection in immunodeficient mice
The immunomodulating and murine cytomegalovirus (MCMV)-inhibiting effects of the synthetic lipid A subunit analogue GLA-60 were investigated in different strains of immunodeficient mice. Peritoneal natural killer (NK) cells obtained from nude (nu/nu) C57BL/6 mice or normal NMRI mice, which had been treated intraperitoneally with 10 µg of GLA-60 1 day earlier, exhibited a greater cytolytic activity than those from untreated mice. GLA-60 also stimulated NK cell activity in SCID (severe combined immune deficiency) mice (which are T and B cell-defective), but not in NK cell-defective beige (C57BL/6 bg/bg) mice. GLA-60 also enhanced the phagocytic activity of peritoneal macrophages in beige, nude and NMRI mice, but not in SCID mice. GLA-60, when administered as a single 150 µg dose 1 day before infection, completely protected beige mice against MCMV-associated mortality. It also caused a significant increase in the life-span of MCMV-infected nude and SCID mice.
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S-Adenosylmethionine metabolism in herpes simplex virus type 2-infected cells
More LessInfection of primary rat embryo cells with herpes simplex virus type 2 has previously been reported to produce a dramatic and rapid inhibition of cellular DNA methylation. However, it has neither an immediate effect on S-adenosylmethionine breakdown nor on the relative pool sizes of S-adenosylmethionine and S-adenosylhomocysteine.
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Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines
More LessVaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybird protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.
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The active adenovirus protease is the intact L3 23K protein
More LessThe L3 23K protein was isolated from adenovirus type 2 and shown to cleave purified substrates, confirming that this protein is the adenovirus protease. Separate antisera, prepared against the amino- and carboxyterminal regions of the 23K protein react with active protease, demonstrating that, contrary to previous reports, zymogen activation is not involved in the regulation of this enzyme. Molecular exclusion chromatography indicated that the protease is active as a monomer. Purified protease was shown to be inhibited by Zn2+ and Cu2+ and by some, but not all, recognized cysteine protease inhibitors, indicating participation of a thiol group and providing additional support to the suggestion that regulation of the enzyme involves a form of thiol-disulphide interchange.
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Neutralization and fusion inhibition activities of monoclonal antibodies specific for the S1 subunit of the spike protein of neurovirulent murine coronavirus JHMV c1-2 variant
More LessThe cleavage products of the spike (S) protein, the S1 and S2 subunits, of the highly neurovirulent murine coronavirus (MHV) JHMV c1-2 variant were identified by immunoprecipitation of virus-infected cell lysates after treatment with urea and 2-mercaptoethanol. By this method 14 monoclonal antibodies (MAbs) raised against the S protein of the c1-2 variant were revealed to react with the S1 subunit and one with the S2 subunit. These 14 MAbs were classified into the following three groups: (A) MAbs reactive to almost all MHV strains examined, (B) MAbs specific for the JHMV strain and (C) MAbs specific for a large S protein of the JHMV strain. All five MAbs classified in group B showed neutralization activity and four of them also showed fusion inhibition activity. Four of six MAbs in group C showed neutralizing activity to the c1-2 variant but not to the sp-4 variant, and most of them had no fusion inhibition activity. Western blot analyses showed that all of the MAbs, except for no. 2 in group A, failed to react with the denatured S and S1 proteins. All MAbs in groups A and C, with the exception of no. 19 in group A, reacted with the mildly denatured S proteins, whereas none of the MAbs in group B did. These results suggest that MAbs in group B recognized highly conformational epitopes which may be involved in the binding of virions to cellular receptors and the fusion activity of the virus.
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Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection
The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoterbased vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.
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Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5′ untranslated region
The nucleotide sequence of the cytopathic Osloss isolate of bovine viral diarrhoea virus (BVDV) was deduced from overlapping cDNA clones and from PCR products. The Osloss genome is an RNA molecule of positive polarity containing 12480 nucleotides and having the capacity to code for a polyprotein of 3975 amino acids. The presence of the previously described internal stop codon in this viral sequence was disproved after direct sequencing of the appropriate PCR-amplified fragment. Except for the previously reported insertion of a sequence coding for a ubiquitin-like protein, the viral genome shares great similarity with those of three other strains of the pestivirus genus. Computer-assisted sequence analyses and comparisons of known pestiviral genomic sequences led us to identify selected PCR primers in the 5′ untranslated region. These primers were used successfully to amplify 18 distinct pestivirus isolates and potential DNA probes were noted from the deduced sequences. The possible use of a well conserved 26 base fragment as a diagnostic probe was confirmed in hybridization experiments. The 5′ untranslated region was further studied and compared with those of other members of the Flaviviridae family, which includes the flaviviruses and the hepatitis C virus group. These sequence analyses support the possibility of discrimination amongst the closely related ruminant pestiviruses, border disease virus and BVDV.
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Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures
The gene encoding the major nucleocapsid, N, protein of measles virus has been inserted into a baculovirus vector under the control of the polyhedrin promoter. Insect cells infected with this recombinant baculovirus synthesize high levels of measles N protein, up to 40% of total soluble cell protein. The recombinant protein is recognized by sera from convalescent patients, vaccinees and patients with subacute sclerosing panencephalitis and thus could form the basis of a simple diagnostic assay. Nucleocapsid-like structures, similar to those found in mammalian cells infected with measles virus, can be observed in both the nucleus and cytoplasm of the infected insect cells. These have many structural features in common with nucleocapsids found in measles virus-infected cells, but are longer (up to 2 µm) and have a lower buoyant density. Measles N protein thus appears to be capable of assembling into nucleocapsidlike structures in the absence of measles virion RNA or other viral proteins.
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Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus
More LessPrevious work has demonstrated that the small hydrophobic (SH) protein of human respiratory syncytial virus (RSV) A2 strain is a 64 amino acid integral membrane protein that accumulates intracellularly as an unglycosylated major species (SH0), a minor species truncated at the amino terminus and two N-glycosylated species one of which contains a further addition of polylactosamine. In this study, the membrane orientation of SH0 was mapped by trypsinization of intact RSV-infected cells followed by washout, lysis and immunoprecipitation of protected fragments with antisera specific for the protein termini. This showed that the C terminus is extracellular and the SH protein was not detectably palmitylated. Analysis of the SH protein by sedimentation on sucrose gradients showed that it rapidly assembles into a homo-oligomer that cosediments with the F protein tetramer. Interestingly, all forms of the SH protein were found in the oligomeric fraction. Chemical cross-linking generated species which appeared to represent dimers, trimers, tetramers and pentamers as well as a minor species of 180K which might correspond to the oligomeric form detected by sucrose gradient sedimentation.
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Studies on a species-specific epitope in murine, ovine and bovine prion protein
More LessTransmissible spongi form encephalopathies are fatal neurodegenerative disorders which are linked to abnormal isoforms of the prion protein (PrP), which is expressed in different cells of various mammalian species. Susceptibility to disease and reduced transmission rates upon the first passage to another species are thought to be a result of functional and biochemical differences of the PrP as a consequence of amino acid sequence among species. In 1985 an epidemic of bovine spongiform encephalopathy (BSE) started after accidential transmission of scrapie by feeding infected sheep and goat meat and bone meal products to cattle. In this report we present data demonstrating speciesspecific epitopes in bovine, ovine and murine PrP that are based on amino acid substitutions at positions 108 and 110. Rabbit antisera to synthetic peptides representing amino acid sequence 108 to 123 of PrP of cattle, sheep and mice reacted strongly with modified PrP of the homologous host but not, or only poorly, with PrP of heterogeneous origin. Cross-reactivity was observed, however, with antisera to bovine and ovine peptide sequences 102 to 117, thus stressing the importance of the location of the amino acid substitution in synthetic peptides used for immunization. Based on these data, BSE PrP and ovine and murine scrapie PrP can be distinguished from each other, and these differences might help elucidate the species barrier effect.
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Replication and morphogenesis of Amsacta moorei entomopoxvirus in cultured cells of Estigmene acrea (salt marsh caterpillar)
More LessA study of the sequence of morphogenic events occurring within Amsacta moorei entomopoxvirus-infected Estigmene acrea cells is presented. Stages in virion development, and the various cytopathic effects observed in these cells between 0 and 120 h post-infection (p.i.) are described. Events in the early stages of virion assembly (24 to 48 h p.i.), the formation of the outer viral membrane (48 to 72 h p.i.) and the development of occlusion bodies or spheroids (72 to 120 h p.i.) were identified. Cells grown in TC100 culture medium supported production of mature virus particles, the majority of which were either of the intracellular naked virion form, or mature virions incorporated into occlusion bodies. Only limited production of the extracellular enveloped form was observed in these cells.
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Close similarity between genome structures of rice black-streaked dwarf and maize rough dwarf viruses
More LessThe complete nucleotide sequences of rice black-streaked dwarf virus (RBSDV) genome segments 8 (S8) and 7 (S7) were determined, and were found to have high sequence identities to the corresponding maize rough dwarf virus (MRDV) genome segments. RBSDV S8 and S7 consisted of 1927 and 2193 nucleotides, respectively. RBSDV S8 had a single long open reading frame (ORF) encoding 591 amino acids. RBSDV S7 had two nonoverlapping ORFs encoding 362 (ORF1) and 309 (ORF2) amino acids. The two ORFs of RBSDV S7 were inserted separately into an Escherichia coli expression vector (pKK223-3). When they were expressed in E. coli cells, the products of both ORFs migrated identically at an apparent M r of 40K. High nucleotide sequence identity was observed between RBSDV S7 and MRDV S6 (85%), and between the terminal regions of RBSDV S8 and MRDV S7. In addition, RBSDV S7 and MRDV S6 showed 91% (ORF1 product) and 85% (ORF2 product) amino acid sequence identities.
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Tobamovirus helper specificity of satellite tobacco mosaic virus involves a domain near the 5′ end of the satellite genome
More LessThe molecular basis of the interactions between plant virus satellites and their helper viruses is not understood. The features of the satellite tobacco mosaic virus (STMV) genome that determine tobamovirus helper specificity were investigated using two independent strategies. The first tested the possible significance of regions of nearly identical sequence within the 3′- terminal 150 bases of the genomes of STMV and its natural helper virus, tobacco mild green mosaic virus (TMGMV). A chimeric STMV clone containing the 3′ terminus of tobacco mosaic virus (TMV-U1) RNA was infectious in coinfections with TMGMV, but it did not replicate with TMV-U1. In the second strategy, populations of STMV adapted to replication with four alternative helper tobamoviruses were generated by serial passage in tobacco. RNase protection analyses of these RNA populations showed that in all cases there had been a genetic change 50 to 60 bases from the 5′ terminus of the STMV genome. Similar changes were detected in several progenies of STMV clones replicated with TMV-U1, indicating that change at this site was essential for replication with a helper virus other than TMGMV. Sequence analyses of the changes at this ‘helper adaptation domain’ showed consistently the deletion of a single G from five consecutive Gs at bases 61 to 65. Infectivity experiments with STMV clones containing this G deletion showed that this change alone was not sufficient to modify helper specificity, so additional factors which remain to be identified must also be involved. Nevertheless, these experiments show the ability of STMV populations to undergo rapid, reproducible evolution by both selection of pre-existing variants and de novo mutation, and constitute the first molecular demonstration that the helper virus acts as a selection pressure on the evolution of satellite populations.
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Signal for potyvirus-dependent aphid transmission of potato aucuba mosaic virus and the effect of its transfer to potato virus X
More LessA British isolate of potato aucuba mosaic potexvirus (PAMV) was transmitted by aphids (Myzus persicae) which had fed previously on a source of potato Y potyvirus (PVY). Nucleotide sequence analysis of the PAMV coat protein gene indicated that amino acid residues 14 to 16 from the N terminus of the coat protein have the sequence DAG, which is also found in the coat proteins of potyviruses and is required for their aphid transmissibility. A recombinant virus isolate (TXPA7) was produced in which a segment of the coat protein gene of PAMV encoding the 40 N-terminal amino acids was inserted in the genome of potato X potexvirus (PVX) in place of the segment encoding the 28 N-terminal amino acids of PVX coat protein. This isolate, and a second similar recombinant (TXPA5) in which the DAG motif was changed to YTS, were mechanically transmissible to intact plants, in which they caused slightly milder symptoms than PVX. Particles of TXPA7 reacted in immunosorbent electron microscopy with PVX- and PAMV-specific antibodies and so were antigenically distinguishable from PAMV and PVX particles, which reacted only with their homologous antibody, and from TXPA5 particles, which reacted only with the PVX antibody. Recombinant TXPA7 was transmitted by aphids that had already fed on a source of PVY whereas TXPA5 and PVX were not. TXPA7 was not transmitted by aphids that had not fed on a PVY source. It is concluded that (i) the potyvirus-dependent aphid transmissibility of PAMV results from possession of a domain which includes the DAG motif and is located near the N terminus of the virus coat protein, and (ii) potyvirus-dependent aphid transmissibility can be conferred on PVX, a non-aphid-borne potexvirus, by substituting this domain for the N-terminal part of its coat protein.
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Efficient production of human gamma interferon in tobacco protoplasts by genetically engineered brome mosaic virus RNAs
More LessWe succeeded in producing human gamma interferon (IFN-γ) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coat protein gene with the IFN-γ gene and synthesized BMV–IFN-γ chimera RNAs using an in vitro transcription system. The BMV–IFN-γ chimera RNAs were used to inoculate tobacco protoplasts together with BMV RNA 1 and RNA 2 and produced IFN-γ to a level of 5 to 10% of total extracted proteins per infected protoplast after 24 h of incubation. The efficient production of IFN-γ was attributed to the high translation activity of the BMV–IFN-γ chimera RNA. We demonstrate that 24 nucleotides coding for the N-terminal amino acids of the full-length coat protein were probably involved in the high translation activity of the BMV–IFN-γ chimera RNA.
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RNA-2 of tobacco rattle virus encodes the determinants of transmissibility by trichodorid vector nematodes
More LessPseudorecombinant isolates produced from an efficiently transmitted and a non-transmitted isolate of tobacco rattle tobravirus were tested for their transmissibility by trichodorid vector nematodes. Isolates with RNA-2 originating from the non-transmissible isolate were not transmitted by the vector, whereas isolates with RNA-2 originating from the efficiently transmitted isolate were transmitted efficiently. It is therefore concluded that the factor determining vector transmissibility is located on the RNA-2 genome segment of TRV. Because the serological properties of the isolates also correlate with transmissibility, it is likely that the virus particle protein is involved in the transmission process.
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Nucleotide sequence of beet cryptic virus 3 dsRNA2 which encodes a putative RNA-dependent RNA polymerase
More LessThe nucleotide sequence of a DNA copy of beet cryptic virus 3 double-stranded RNA2 was determined, and one strand was found to contain a single long open reading frame of 1431 nucleotides which encoded a putative polypeptide containing 478 amino acid residues with an M r of 54·9K. This polypeptide contained conserved amino acid sequence motifs found in the genes that encode putative RNA-dependent RNA polymerases of other RNA viruses.
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The multiplication in plants of arabis mosaic virus satellite RNA requires the encoded protein
More LessOligonucleotide-directed mutagenesis was used to create two mutations at each of three positions within the open reading frame (ORF) of a cDNA clone representing a satellite RNA from a lilac isolate of arabis mosaic nepovirus (ArMV). Three of the six mutants, in which stop codons were introduced at three different sites, did not direct synthesis of a translation product. The other three mutants, in which stop codons were not introduced, directed synthesis of a translation product (39K) although, in two of these, the mutation led to a single amino acid substitution. When Chenopodium quinoa plants were inoculated with in vitro transcripts from each of the six mutants together with the genomic RNA molecules (RNA-1 and RNA-2) of ArMV, progeny RNA was detected only with two of the three mutants in which the nucleotide changes did not introduce a stop codon to the coding region. To look for complementation, two deletion mutants were made. In these, 113 or 117 nucleotides were removed from two consecutive regions within the ORF. Two insertion mutants (in which the deleted sequences were replaced with a 130 nucleotide sequence from RNA-2 of cherry leaf roll nepovirus) were also made. Transcripts from none of these mutants retained messenger activity and none was detected either in C. quinoa plants or in virions, even in the presence of wild-type satellite RNA.
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