- Volume 78, Issue 7, 1997
Volume 78, Issue 7, 1997
- Articles
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Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma
Hepatitis C virus (HCV) exists in vivo as a highly variable mixture of closely related genomes (quasispecies), but the pathogenetic significance of such heterogeneity is still largely unknown. To investigate this issue, we compared the composition of HCV quasispecies found in the liver, peripheral blood mononuclear cells (PBMC) and plasma of ten patients by single-strand conformation polymorphism analysis of the E2/NS1 region and sequencing of the variants detected. We found considerable quasispecies differences between the liver and PBMC in all the patients, involving variant numbers, relative quantities and relative electrophoretic mobilities, but no apparent tissue-specific trend. Genome variants present in the liver and/or PBMC were not detected in the corresponding plasma samples, while certain HCV variants were present only in plasma. No dominant amino acids or amino acid pattern characteristic of variants present solely in the PBMC were detected in the E2/NS1 region sequenced.
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Hepatitis C virus core protein induces hepatic steatosis in transgenic mice
Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, which finally leads to development of hepatocellular carcinoma. Chronic hepatitis C is characterized by several histological features in the liver which discriminate it from other forms of hepatitis: bile duct damage, lymphoid follicles and steatosis (fatty change). Little is known, however, about the role of HCV or its viral proteins in the pathogenesis of hepatitis. Recently, the core protein of HCV has been suggested to have a transcriptional regulatory function, and thereby to be involved in inducing phenotypic changes in hepatocytes. To clarify whether or not the HCV core protein has an effect on pathological phenotypes in the liver, two independent transgenic mouse lines carrying the HCV core gene were established. These mice developed progressive hepatic steatosis, indicating that the HCV core protein plays a direct role in the development of hepatic steatosis, which characterizes hepatitis C. This transgenic mouse system would be a good animal model for the study of pathogenesis in human HCV infection.
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Discrimination of hepatitis G virus/GBV-C geographical variants by analysis of the 5' non-coding region
We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5′ non-coding region (5′NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5′NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaire and Scotland, pro duced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5′NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.
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Secondary structure of the 3'-untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development
More LessA genetic algorithm-based RNA secondary structure prediction was combined with comparative sequence analysis to construct models of folding for the distal 380 nucleotides of the 3′-untranslated region (3-UTR) of yellow fever virus (YFV). A number of structural elements that are thermodynamically stable, conserved in shape, and confirmed by compensatory mutations were revealed. At the same time structural polymorphisms were observed among strains of YFV. These polymorphisms showed an association with virulence: all wild and pathogenic strains were likely to be folded in a signifi-cantly different way from vaccine strains with reduced virulence. Structural divergence was also found among vaccine strains, with 17DD, the most virulent in the mouse model, exhibiting an intermediate pattern of folding, combining structural features of both wild and vaccine strains. The observation of a strong association between secondary structure of the 3′-UTR and virulence of YFV may help elucidate the molecular mechanisms of virus attenuation and lead to new strategies of vaccine development directed towards rational modification of secondary structure of the 3′-UTR.
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Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus
The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5′ nontranslated region has two nucleotide changes, and the 3′ nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and nonstructural regions of the SFV genome.
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Death mechanisms in cultured cells infected by Semliki Forest virus
More LessWe have investigated the induction of cell death in cultured cells by the virulent SFV4 and avirulent A7 strains of Semliki Forest virus (SFV). In BHK cells, death occurred by a typical apoptotic mechanism, as did the death of oligodendrocytes in glial cell cultures. For cerebellar neuron cultures, virus- induced death was due to necrosis. Although the SFV4 and A7 strains did not differ in the mechanism of induction of cell death, the virulent SFV4 strain did multiply to a higher titre in cultured neurons than the avirulent A7 strain. This is consistent with previous animal studies which indicate that the virulence of SFV strains is controlled by rapidity of multiplication in the CNS, leading to a lethal threshold of damage, rather than differential cell tropism or cell death mechanisms. The immune- mediated demyelination induced by avirulent strains may be triggered by apoptosis of oligodendrocytes, the consequences of which are obscured by death for virulent strains.
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Molecular cloning of the hepatitis A virus receptor from a simian cell line
More LessUsing a eukaryotic expression system in combination with a monoclonal antibody (MAb) capable of blocking hepatitis A virus (HAV) adsorption, a cDNA clone was selected from a library of S.la/Ve-1 cells, a cell line that is highly susceptible to the virus. Sequence analysis of the cDNA revealed a single open reading frame that encoded a protein consisting of 460 amino acids. The deduced primary structure of the protein included a signal sequence, a transmembrane domain, four sites for ^-linked glycosylation, cysteine residues attributable to an immunoglobulin domain and threonine clusters characteristic of mucin-like protein. By employing a vaccinia virus expression vector, the cDNA was expressed in HeLa cells where it induced marked HAV attachment which was specifically blocked by the MAb. The cDNA obtained was thus assumed to encode a functional receptor for HAV.
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The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase
More LessThe genome sequence of the rabies-related virus Mokola virus (genus Lyssavirus) has been completed by sequencing the L gene, which consists of 6384 nucleotides encoding a 2127 amino acid polymerase. Alignment of the Mokola virus L protein with other polymerases from the virus order Mononegavirales defined three domains: a divergent NH2-terminal domain, a highly conserved central domain carrying most of the functional motifs and a COOH- terminal domain with alternating conserved and divergent regions. A statistical study outlined the stringency of conservation of glycine, acidic (D, E) and basic (K, R, H) amino acids in polymerases, particularly as key residues of the conserved motifs.
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Class I-restricted CTL induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin
More LessWe have investigated the class I-restricted CTL response specific for measles virus haemagglutinin (HA) in the spleens of mice immunized by various mucosal routes with a DNA plasmid carrying the HA gene (pV1j-HA). A single immunization with recombinant DNA injected in the buccal mucosa induced an HA-specific CTL response. Similarly, nasal immunization with the DNA vaccine induced primary CTLs against measles virus HA. Booster immunization did not enhance the CTL activity. Oral or intrajejunal immunization with the plasmid induced a CTL response of lower magnitude. However, this could be potentiated by co-administration of the mucosal adjuvant cholera toxin or cationic lipids (DOTAP). These data show that a CTL response can be generated by mucosal vaccination using DNA vaccines.
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Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype
More LessSequence analysis was performed on 20 measles virus (MV) isolates from South Africa, five of which were obtained between 1986 and 1989 and 15 isolates collected during the 1994/95 measles season. A 590 bp fragment of the carboxyl terminus of the nucleocapsid (N) was amplified by PCR and subjected to sequence and phylogenetic analysis. Comparison of the South African MV strains with those previously described revealed that at least two distinct groups of wild-type (wt) MV exist, one of which has been circulating since 1986. The major genotype (I) was represented by the more recent isolates which showed three characteristic amino acid substitutions. Furthermore, three vaccine-like viruses with sequences very similar to the Edmonston wt strain were identified. Phylogenetic analysis of 100 MV strains allowed the assignment of new definitions for MV genotypes and subgroups. Employing these definitions, the majority of South African isolates analysed here formed a new genotype.
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Influenza virus M1 protein binds to RNA through its nuclear localization signal
More LessThe RNA-binding activity of influenza A virus M1 protein was studied by cross-linking the protein to viral RNA followed by sequence analysis of the oligoribonucleotide bound to the protein as well as sequence analysis of the M1 peptide bound to the RNA. M1 was found to bind to RNA without any RNA sequence specificity, as verified in a series of filterbinding experiments using a large variety of nucleic acids including DNA. The peptide sequence that bound to the RNA was the RKLKR nuclear localization signal of M1. Site-specific mutagenesis of recombinant M1 showed that most of the basic residues in that region had to be mutated in order to inhibit RNA-binding. We also constructed an M1 mutant that no longer bound to RNA but which was still able to inhibit the in vitro transcription activity of isolated viral ribonucleoprotein, albeit to a lower extent. Mutation of the zinc-binding sequence had no effect on RNA-binding or transcription-inhibition activity.
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Role of gamma delta TCR+ lymphocytes in the augmented resistance of trehalose 6,6'-dimycolate-treated mice to influenza virus infection
More LessTrehalose 6,6′-dimycolate (TDM), an immunomodu- lator, potentiates non-specific resistance in mice to influenza virus infection. When mice were injected intravenously with TDM, the striking proliferation of a minority of T-lymphocytes bearing gamma/delta T-cell receptors (γδ T-cells) that accumulated in granulomatous lungs was thought to be associated with the maintenance of acquired resistance to lethal influenza virus infection. To clarify the cellular basis of the defence against influenza virus, mice were depleted of γδ T-cells, alpha/beta (ap) T-cells, or natural killer (NK) cells by in vivo administration of corresponding antibodies prior to influenza virus infection. The depletion of γδ T-cells significantly abrogated the augmented resistance of TDM- treated mice to infection, as did depletion of either αβ T-cells or NK cells. To gain insight into the functional ability of γδ T-cells, we evaluated the cytotoxic activity ofthisT-cell subset againsta panel of target cell lines that were stably transfected with the influenza virus haemagglutinin (HA) gene from A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) strains. The γδ T-cells from TDM-treated mice showed profound cytotoxicity against the target cells expressing HA of either the H1 or H3 subtype, in a non-major histocompatibility complex-restricted manner. Taken together, these results indicate that γδ T-cells play a non-specific role, in conjunction with αβ T-cells and NK cells, in protecting mice against influenza virus infection, and that the recognition and destruction of HA-expressing target cells by the activated γδ T-cells is one of the steps involved in this anti-influenza virus immuno- surveillance.
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Molecular characterization of attenuated Junin virus strains
The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.
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African horsesickness virus VP7 sub-unit vaccine protects mice against a lethal, heterologous serotype challenge
More LessAn established mouse model was used to evaluate the effectiveness of the major outer core protein of African horsesickness virus (AHSV), VP7, as a subunit vaccine. Adult female BALB/c mice were immunized with VP7 crystals purified from BHK cells infected with AHSV serotype 9 (AHSV-9), using three inoculations in Freund’s adjuvant. Eighty to one hundred per cent of the immunized mice were protected against a heterologous challenge with a known lethal dose of AHSV-7. The protected immunized mice did not develop any clinical signs characteristic of virulent AHSV infection in this model during the study. In contrast, 80–100% mortality was observed in the non-immunized mice that received the same challenge virus. Subsequent studies indicated that a single inoculation of 1·5 µg purified AHSV VP7 in Freund’s complete adjuvant was sufficient to protect at least 90% of mice from AHSV-7 challenge. If the antigen was presented in the absence of Freund’s complete adjuvant, 70% of the mice were still protected by one inoculation of VP7 crystals. Titres of circulating antibody against AHSV VP7, determined by competitive ELISA, did not appear to correlate with protection and passive antibody transfer from immunized BALB/c mice failed to protect syngeneic recipients from AHSV-7 challenge. Therefore, the observed protection is unlikely to be due to an antibody-mediated immune response. The number of viraemic mice and the duration of viraemia post-challenge was significantly reduced in vaccinated mice compared to non- vaccinated controls. However, the levels of viraemia were similar.
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VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis
More LessThe importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overlay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM2 insect cells.
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Differential T cell response induced by certain recombinant oligopeptides of herpes simplex virus glycoprotein B in mice
More LessMuch attention is presently focused on the quality of the immune response produced by helper T or regulatory cells because of its implications for vaccine development and immunomodulation. Glycoprotein B (gB) of herpes simplex virus (HSV) has been shown to induce a protective T cell response. To further characterize the nature of the T cell response, oligopeptides were expressed from the open reading frame of gB from HSV-2 (gB-2) as fusion proteins with β-galactosidase (GZ) in E. coli. After immunopurification using an anti-GZ affinity column, oligopeptides p59 and p65, spanning amino acid residues 339-394 and 424-484 of gB- 2 respectively, were examined for immunogenic response by delayed type hypersensitivity (DTH) in vivo and for antigenic response by T cell proliferation in vitro. p59 but not p65 was able to prime for both DTH and proliferative T cell response to whole HSV-2 and protect against challenge infection. However, when mice were pretreated with cyclophosphamide, p65 primed for a strong DTH response to a level similar to that induced by p59 in mice either pretreated or not treated with cyclophosphamide. This suggests that p65 contains epitopes capable of inducing both DTH and immunosuppression. Thus, when mice were primed with p65 before immunizing with HSV-2, their in vitro HSV- specific proliferative response was suppressed. Therefore, p59 is a good immunogen able to induce significant, though incomplete, protection. It could be considered for inclusion in a cocktail of subunit vaccines against HSV-2 whereas p65 or parts thereof should be excluded for this purpose.
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Efficient herpes simplex virus type 1 (HSV-1) capsid formation directed by the varicella-zoster virus scaffolding protein requires the carboxy-terminal sequences from the HSV-1 homologue
More LessThe scaffolding protein and associated protease of the human herpesvirus varicella-zoster virus (VZV), encoded by genes 33·55 and 33 respectively, were synthesized in insect cells using a baculovirus expression system. The expressed 33·55 product formed numerous long, flexible, hollow rods, and in this respect differed from the herpes simplex virus type 1 (HSV-1) homologue which forms large aggregates consisting mainly of fibrous material interspersed with scaffold-like particles. Removal of 27 amino acids from the carboxy terminus of the VZV scaffolding protein by the gene 33 protease or expression of the cleaved product did not result in any discernible change in the morphology of the scaffolding protein. Again, this was in marked contrast to the situation in HSV-1 where removal of the 25 carboxy-terminal amino acids from the scaffolding protein by the associated protease or expression of VP22a results in the formation of large numbers of scaffold-like particles. Despite these differences, when cells were multiply infected with baculoviruses expressing the HSV-1 capsid shell proteins and the VZV scaffolding protein complete capsids were observed, suggesting that the VZV protein could act as a scaffold for the assembly of the HSV-1 capsid shell. The efficiency of capsid assembly was increased substantially by exchanging the 23 carboxy-terminal amino acids of the VZV scaffolding protein for the corresponding 22 carboxy-terminal amino acids of the HSV-1 homologue, supporting previous work which showed that this region was critical for the formation of intact capsids.
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Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55)
More LessTo define structural elements involved in translocation of human cytomegalovirus (HCMV) glycoprotein B (gB) to the inner nuclear membrane (INM) compartment, mutagenized gB derivatives with deletions of the potential membrane anchor domains or of portions of the cytoplasmic tail were stably expressed in human astrocytoma cells. Subcellular localization examined by immunofluorescence and cell fractionation suggested that all gB derivatives reached the INM; however, reduced amounts were found after deletion of the extreme carboxy terminus [amino acids 856–906; gB(Del3)]. Pulse-chase analysis revealed accumulation in nuclear fractions of all gB derivatives during the chase, except for gB(Del3), which exhibited impaired nuclear retention. A carboxy-terminal nucleoplasmin-like signal localized within the respective deletion may thus be involved in nuclear transport and retention of HCMV gB. Immunoprecipitation after 32P-radiolabelling of the gB transfectants verified that the gB molecule is phosphorylated at a carboxy-terminal consensus motif for casein kinase II.
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Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression by adenoviral vectors
More LessWe have developed a number of replication defective adenoviral (Ad) vectors which express transgenes under the control of the human cytomegalovirus (HCMV) immediate early (IE) gene promoter. The orientation of the expression cassette replacing E1 in the vector backbone had a significant effect on the level of transgene expression, with vectors containing expression cassettes directed towards the right end of the Ad genome expressing 7-fold higher levels of β-galactosidase (β-gal) than those with inserts in the opposite orientation. Murine cells infected with any of several different Ad vectors in which transgene expression was under the control of the HCMV IE promoter produced 10–100-fold less transgene product (such as β-gal) than similarly infected human cells. Replacing the HCMV IE promoter with the murine CMV (MCMV) IE promoter resulted in an increase in the levels of β-gal produced in murine and rat cells by approximately 5–30-fold compared to levels obtained with the HCMV IE promoter, and levels produced in human cells were the same or greater using the MCMV IE promoter compared to the HCMV IE promoter. Similar results were obtained using a luciferase reporter gene. The MCMV IE promoter, therefore, was able to drive high levels of expression without the pronounced species preferences observed for the HCMV IE promoter. The MCMV IE promoter also directed high levels of expression in vivo, suggesting that Ad vectors carrying the MCMV IE promoter may be more effective than those with the HCMV IE promoter for transgene expression in animal models.
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Sequence variations in EBNA-1 may dictate restriction of tissue distribution of Epstein-Barr virus in normal and tumour cells
In seropositive individuals Epstein-Barr virus (EBV) establishes a virus reservoir in peripheral blood lymphocytes (PBLs). Transmission from one individual to another occurs via saliva due to a lytic (virion productive) phase of infection in the oropharynx. EBNA-1 is responsible for maintaining viral episomes in the host cell and could, therefore, also affect the persistence of the virus in different cell lineages. Based on sequence analysis of EBNA-1 we now demonstrate that (i) in addition to the prototype EBNA-1 (identical to the B95.8 virus EBNA-1), EBV in normal individuals encompasses multiple EBNA-1 subtypes, both in PBLs and in oral secretions; (ii) although EBV with prototype EBNA-1 is the predominant virus in normal individuals, it is very rarely associated with either nasopharyngeal carcinoma (NPC) or Burkitt’s lymphoma (BL); (iii) EBV with an EBNA-1 subtype (V-val) frequently associated with NPC is also selectively detected in oral secretions and not in PBLs; (iv) EBV with the EBNA-1 subtype V-pro is restricted to PBLs, while a mutated version of this subtype is present in BL, but not in NPC. These findings suggest that the variations in EBNA-1 may be relevant to the ability of EBV to persist in different cell types, and hence relevant to its oncogenic potential.
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Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N
More LessUsing the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-Jκ. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A. The region of RBP-2N to which EBNA6A binds has previously been shown to interact with EBNA2.
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Murine gammaherpesvirus 68 encodes tRNA-like sequences which are expressed during latency.
More LessMurine gammaherpesvirus 68 (MHV-68) is a virus of wild rodents and is a convenient small animal model for studies of gammaherpesvirus pathogenesis. We have sequenced 6162 bp at the left end of the MHV-68 genome and identified two unique open reading frames (ORFs) (ORF2 and ORF3) and an ORF (ORF1) which displays similarity to poxvirus members of the serpin family. Interspersed with the ORFs is a family of eight novel tRNA-like sequences sharing tRNA-like predicted secondary structures and RNA polymerase III promoter elements. These sequences are expressed to high levels during lytic infection and are processed into mature tRNAs with post-transcriptionally added 3′ CCA termini, indicating their recognition as tRNAs by cellular machinery. Acidic Northern analysis of four tRNAs tested has demonstrated that they are not amino-acylated by aminoacyl-tRNA synthetases present in the infected cell. Thus, it is currently unclear what biological function these uncharged viral tRNA-like sequences may fulfil. In situ hybridization analysis has shown that in addition to being expressed within productively infected tissues during acute stages of infection, the tRNA-like sequences are abundantly expressed within splenic germinal centres of latently infected mice. Therefore, the MHV-68 viral tRNAs represent a marker for latent infection and constitute the first report of tRNA-like sequences encoded by a virus of eukaryotes.
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Specificity of human cytotoxic T lymphocytes induced by a human papillomavirus type 16 E7-derived peptide
In order to establish tumour-specific cytotoxic T lymphocyte (CTL) cell lines, T cells from a human papillomavirus (HPV) type 16-positive patient with a cervical carcinoma in situ and from a healthy volunteer were stimulated in vitro with autologous dendritic cells loaded with peptides derived from the viral transforming proteins E6 and E7 and corresponding to potential HLA-A*0201-restricted T cell epitopes. From each donor a small number of low-affinity CTL lines against the peptide E7/86–93 was obtained, which specifically lysed HLA-A*0201-expressing B-lymphocytes (cell line 721) loaded with this peptide. Cytotoxicity was also observed against two HLA-A*0201-E7-positive epithelial cell lines, the cervical carcinoma cell line CaSki and the HPV-16-immortalized foreskin-keratinocyte cell line HPK IA. However, since none of the CTL recognized both cell lines, and E7-expressing 721 transfectants were never lysed, it was concluded that the reactivity against CaSki and HPK IA cells was due to cross-reactivity on allogeneic HLA molecules rather than to E7 recognition, which emphasizes that the specificity of tumour cell lysis by peptide-induced CTL has to be interpreted with caution.
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Tissue culture adaptation of natural isolates of simian virus 40: changes occur in viral regulatory region but not in carboxy-terminal domain of large T-antigen.
More LessThe regulatory region of natural isolates of simian virus 40 (SV40) is different from that of laboratory-adapted strains of the virus. The latter have a nucleotide sequence duplication within the enhancer region which varies slightly with each strain, whereas the duplication is lacking in fresh isolates of SV40, which contain an ‘archetypal’ regulatory region. Many isolates also display nucleotide differences in the DNA encoding the carboxy terminus of large tumour antigen (T-ag). To determine whether genetic changes in these two regions of the SV40 genome were detectable during laboratory adaptation and long-term passage, low-passage virus stocks of two laboratory strains which had detailed passage histories spanning more than 25 years (Baylor strain and VA45-54) were analysed using PCR, cloning and sequencing assays. Both laboratory and archetypal regulatory regions were present in low-passage stocks. Following duplication in the regulatory region, no additional changes were detectable. The variable region at the T-ag carboxy terminus did not undergo any change with tissue culture passage and may serve as a useful site for taxonomic classification of different strains of SV40. Cloned genomes containing single or duplicated enhancers derived from both SV40 strains were viable in CV-1 cells. Attempts to induce regulatory region duplications by 14 serial passages of SV40 archetypal strains in monkey cells were not successful. The results are compatible with tissue culture adaptation of SV40, reflecting either selection of a rare variant pre-existing in the original sample or generation of a rare regulatory region duplication in infected cells.
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Sequence heterogeneity of heron hepatitis B virus genomes determined by full-length DNA amplification and direct sequencing reveals novel and unique features
More LessSo far, only a single heron hepatitis B virus genome (HHBV-4) has been cloned and sequenced. Therefore, neither the significance of its sequence divergence from other avian hepadnaviruses nor the sequence variability of HHBV genomes in general are known. Here we have analysed the sequence heterogeneity of HHBV genome populations in several sera from naturally infected herons. A highly sensitive PCR method for full-length HHBV genome amplification was established which allowed direct sequencing of entire HHBV populations without prior cloning. Sequences of HHBV genomes from four sera were thus obtained which differed from those of HHBV-4 by up to 7%. Some of the divergent nucleotides and the corresponding amino acids of the predicted viral proteins were conserved in all four new HHBV isolates and varied only in HHBV-4. This indicates that the HHBV-4 genome is not in all aspects representative of this class of viruses. Interestingly, a highly conserved ORF upstream of the C-gene present in a position analogous to that of the mammalian hepadnavirus X-gene became apparent in all HHBV genomes. In contrast to the duck hepadnaviruses, the small (sAg-S) instead of the largest (sAg-L) envelope protein of all HHBVs has a myristylation site. These data confirm the significant sequence divergence of HHBV from other avian hepadnaviruses. Moreover, they show that HHBV has low sequence variability and indicate two new and unique features not evident in other avihe-padnaviruses: an additional, highly conserved gene and potential myristylation of the sAg-S instead of the sAg-L envelope protein.
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A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa.
More LessThe preS2/S genes of hepatitis B virus isolated from 29 acutely or chronically infected individuals in the Gauteng province of South Africa were sequenced. Phylogenetic analysis of these sequences in comparison with global isolates from the GenBank database showed that 24 sequences clustered with genotypic group A, three with genotypic group D and one each with genotypic groups B and C. Group A isolates had greater identity with groups D (variation of 6·6%) and E (6·8%) than with the Eastern groups B (7·4%) and C (8·1%) and were most different from group F (11·0%). Of the South African group A specimens, 59·1% clustered with two global sequences to form a discrete segment which we have called subgroup A . The amino acid differences that set these isolates apart from the rest of group Atended to cluster in the preS2 region (amino acids7,10, 32, 35,47,48, 53 and 54), with a few changes occurring in the major surface antigen (amino acid sites 207 and 209). Analysis of isolates showed that there was a 9-fold higher prevalence of the ay determinant in South Africa than previously reported.
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Structure and genomic organization of a novel human endogenous retrovirus family: HERV-K (HML-6).
More LessPrototypic elements of a novel human endogenous retrovirus (HERV) family were identified and cloned from a human genomic library by the use of a pol fragment, HML-6, related to type A and type B retroviruses and class II HERVs. Out of 39 pol-hybridizing clones, five contained structures of full-length retroviral proviruses, with regions showing similarity to gag, pol and env, flanked by long terminal repeats (LTRs). Restriction mapping and partial sequence analysis of each full-length clone revealed few conserved restriction sites among HML-6 genomes, and about 20% sequence divergence over the reverse transcriptase region sequenced, suggesting that HML-6 constitutes a heterogeneous, but distinct family of elements belonging to the HERV-K superfamily. Sequence analysis of two clones, HML-6p and HML-6.17, revealed a lysine (K) tRNA UUU primer-binding site, and 40–68% nucleotide sequence similarity to LTR, gag, pro, pol and env regions of type B retroviruses and class II HERVs. HERV-K (HML-6) elements are present at about 30–40 copies per haploid genome. The HML-6 LTRs contain putative progesterone-responsive elements, which may be involved in the regulation of HML-6 expression. Furthermore, there are about 50 additional solitary HML-6 LTRs per haploid genome. Such LTRs were integrated within the pol region of two clones belonging to the same HML-6 family, indicating that some site preference may be involved in HERV integration.
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Transfer of endoplasmic reticulum and Golgi retention signals to human immunodeficiency virus type 1 gp160 inhibits intracellular transport and proteolytic processing of viral glycoprotein but does not influence the cellular site of virus particle budding.
More LessIn this study, specific signals known to mediate endoplasmic reticulum or Golgi localization of transmembrane proteins have been transferred to the human immunodeficiency virus type 1 (HIV-1) env gene product. The intracellularly retained recombinant glycoproteins were not proteolytically processed to gp120 and gp41, which is further evidence that this process occurs at a later stage in the transport pathway, presumably within or near the trans-Golgi network. Since the subcellular localization of the viral glycoproteins of enveloped viruses can be one of the factors determining the cellular site of particle assembly and release, experiments were performed to determine if this property was altered by coexpression of the recombinant HIV-1 glycoproteins. When wild-type virus was compared to mutant virus encoding the intracellularly retained glycoproteins, the extent of HIV-1 particle release into the extracellular medium remained unaffected, and electron-microscopic analysis did not reveal any significant alteration in the cellular sites of particle assembly and budding. Thus, in COS-7 cells, altered subcellular localization of the viral glycoprotein does not exert a dominant influence on the assembly site of the HIV-1 particle.
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A protoplast system for studying tomato spotted wilt virus infection.
A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.
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A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.
A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza clostero- virus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5′ and 3′ ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0·47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.
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Transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.
More LessAn expression cassette based on the highly specific tobacco etch potyvirus (TEV) nuclear inclusion (NIa) proteinase has been developed to produce multiple proteins through the translation of a single selfprocessing polypeptide. Gene constructs encoding TEV NIa, the tobacco mosaic tobamovirus (TMV) coat protein (CP) and the soybean mosaic potyvirus (SMV) CP were used to develop transgenic tobacco plants. Proper processing of the multifunctional polypeptide was demonstrated, leading to accumulation of separate proteins in pianta. Moreover, the viral genes expressed in this way were biologically active and conferred pathogen-derived protection to TMV, TEV and potato potyvirus Y (PVY). Transgenic plants were also derived from gene constructs in which the NIa cleavage site was mutated, resulting in the accumulation of the non- processed polyprotein, as predicted. Although transgenic proteins accumulated in low amounts in all the plant lines analysed, accumulation of the mutant non-processed protein form was greatly increased in plants following infection with TEV, but not TMV, apparently as a consequence of protein stabilization.
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Detection of potato mop-top virus capsid readthrough protein in virus particles.
G H Cowan, L Torrance and B ReavyPotato mop-top furovirus (PMTV) RNA 3 encodes the 20 kDa coat protein and a larger readthrough protein of 67 kDa. The readthrough protein is expressed by suppression of the amber stop codon which terminates the coat protein gene. A 21 kDa C-terminal fragment of the readthrough protein was cloned, fused to glutathione S-transferase and expressed in E. coli. An antiserum prepared against purified fusion protein was used in ELISA to detect the readthrough protein in extracts of PMTV- infected leaves. Immunogold labelling studies showed that the readthrough protein was located near one extremity of some of the virus particles.
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Nucleotide sequence of a new bipartite geminivirus isolated from the common weed Sida rhombifolia in Costa Rica.
More LessThe nucleotide sequence of infectious clones of a geminivirus from Costa Rica that infects Sida rhombifolia was determined. Sida golden mosaic virus (SiGMV-Co) has a bipartite genome (DNAs A and B). Computer analysis showed that the bipartite genome of SiGMV-Co resembles that of other whitefly-transmitted geminiviruses. The DNA A (2605 nt) and DNA B (2587 nt) components have little sequence homology other than within the common region (CR). Analysis of DNAs A and B showed that SiGMV-Co is closely related to bean dwarf mosaic virus (BDMV). SiGMV-Co was introduced via agroinoculation into seven plant species, including tomato and bean.
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Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components.
More LessClones of two subgroup III geminiviruses, the common strain of tomato golden mosaic virus (csTGMV) and African cassava mosaic virus originating from Kenya (ACMV-K), were shown to be non-transmissible by whiteflies. Lack of trans- missibility of cloned ACMV-K was investigated by exchanging genomic components with a whitefly- transmissible ACMV isolate from Nigeria (ACMV- NOg). Neither pseudorecombinant was transmissible, indicating that defects in both genomic components contributed to the lack of trans- missibility. Analysis of the acquisition of the pseudorecombinants by Bemisia tabaci indicated that accumulation of virus within the insect was DNA B dependent. Return of virus to plants was determined by DNA A, although the coat protein was essential for acquisition. Repeated passaging of both the wild strain of ACMV-NOg and the cloned virus led to loss of insect transmissibility of the wild isolate but not the cloned virus. Products encoded on both genomic components are required for transmission of bipartite geminiviruses by B. tabaci.
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Nicking and joining activity of banana bunchy top virus replication protein in vitro.
More LessThe major open reading frame of banana bunchy top virus (BBTV) DNA-1 encodes a putative replication initiation protein (Rep). In vitro, a fusion protein of BBTV Rep linked to a maltose-binding protein exhibited both site-specific nicking and joining activities. These activities were dependent on the presence of Mg2 or Mn2 , but did not require ATP. The fusion protein specifically cleaved ssDNA between bases 7 and 8 of a conserved nonanucleotide loop sequence which is present in the virion-strand of the stem-loop common region of each BBTV component. During this reaction, the fusion protein became covalently attached to the 5′ end of the 3 cleavage product. After the nicking reactions, the fusion protein was also capable of catalysing the joining of two nicked ssDNA fragments in a site-specific manner. Based on these activities, BBTV Rep would appear to be very similar to the Rep proteins of the geminiviruses.
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Evidence for the presence of a low-level, persistent baculovirus infection of Mamestra brassicae insects.
More LessA laboratory culture of Mamestra brassicae insects (MbLC) harbours a latent or occult baculovirus that resembles M. brassicae multiple nucleocapsid nucleopolyhedrovirus (MbMNPV). Although conventional extraction techniques have failed to detect the presence of virus in MbLC, control virusfree insects (MbWS) died of an MbMNPV-like infection after being fed MbLC fat-body cells. This suggested that the MbLC cells harboured infectious MbMNPV, albeit at low levels. We have also demonstrated that fat-body cells from MbLC, but not from MbWS, contain mRNA specific for the polyhedrin gene and transcriptional factors that are capable of activating baculovirus late and very late gene promoters linked to a reporter gene encoding chloramphenicol acetyltransferase. Our data provide indirect evidence that the latent MbMNPV in the MbLC insects is maintained as a persistent infection, with the expression of viral genes at a low level.
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Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.
More LessPromoter activity of the Campoletis sonorensis polydnavirus (CsPDV) WHv1.6 gene was analysed by transient transfection assays in insect cell culture using constructs expressing the CAT gene. Deletions of the WHv1.6 gene promoter were used to define promoter regions important for expression. Progressive deletion of the regions upstream of the TATA box reduced the promoter activity, whereas deletions eliminating the TATA box abolished promoter activity. Cis-activating elements were detected up to 1 kb upstream of the WHv1.6 transcription initiation site (TIS). Promoter elements increasing transcription were detected between 444 and 550 bp and between 831 and 1035 bp relative to the TIS. Analysis of the 3′ flanking sequences of the WHv1.6 gene indicated that the polyadenylation signals were the only important elements affecting expression in the constructs. Comparison of promoter regions of four cysteine-rich CsPDV genes revealed homologous sequences that may be important for transcriptional regulation of polydnavirus gene expression in parasitized Heliothis virescens larvae.
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