- Volume 82, Issue 4, 2001
Volume 82, Issue 4, 2001
- SGM Special Lecture
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2000 Fleming Lecture. The origin and evolution of hepatitis viruses in humans
More LessThe spread and origins of hepatitis C virus (HCV) in human populations have been the subject of extensive investigations, not least because of the importance this information would provide in predicting clinical outcomes and controlling spread of HCV in the future. However, in the absence of historical and archaeological records of infection, the evolution of HCV and other human hepatitis viruses can only be inferred indirectly from their epidemiology and by genetic analysis of contemporary virus populations. Some information on the history of the latter may be obtained by dating the time of divergence of various genotypes of HCV, hepatitis B virus (HBV) and the non-pathogenic hepatitis G virus (HGV)/GB virus-C (GBV-C). However, the relatively recent times predicted for the origin of these viruses fit poorly with their epidemiological distributions and the recent evidence for species-associated variants of HBV and HGV/GBV-C in a wide range of non-human primates. The apparent conservatism of viruses over long periods implied by these latter observations may be the result of constraints on sequence change peculiar to viruses with single-stranded genomes, or with overlapping reading frames. Large population sizes and intense selection pressures that optimize fitness may be the factors that set virus evolution apart from that of their hosts.
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- Animal: RNA Viruses
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Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3′ end of the hepatitis G virus/GB-virus C genome
More LessHepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787–5794, 1999). In this study we have carried out a detailed comparison of the structure of the 3′untranslated region (3′UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1–4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem–loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.
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Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway
More LessHepatitis C virus (HCV) persists in the majority of infected individuals and is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis C is currently treated with interferon (IFN)-α or with a combination of IFN-α and ribavirin. The availability of an HCV replicon system (Lohmann et al., Science 285, 110–113, 1999) allowed the investigation of the effects of IFN on genuine HCV replication in cultured cells. It is shown here that IFN-α inhibits subgenomic HCV RNA replication in HuH-7 human hepatoma cells. Immunofluorescence, Western blot and Northern blot analysis revealed that levels of both HCV protein and replicon RNA were reduced after treatment with IFN-α in a dose-dependent manner. In further experiments, it was investigated whether MxA plays a role in the inhibition of HCV. The human MxA protein is an IFN-induced GTPase that has antiviral activity against various RNA viruses. However, HCV RNA replication was not affected in transfected HuH-7 cells that transiently overexpressed MxA. Moreover, a dominant-negative mutant of MxA did not interfere with the antiviral activity of IFN-α against HCV RNA replication. Taken together, these results demonstrate that IFN-α inhibits HCV replicons via an MxA-independent pathway.
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A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-β and the viral helicase, NS3
More LessThe dengue virus RNA-dependent RNA polymerase, NS5, and the protease/helicase, NS3, are multidomain proteins that have been shown to interact both in vivo and in vitro. A hyperphosphorylated form of NS5 that does not interact with NS3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of NS5 (residues 369–405). In this study, it is shown by using the yeast two-hybrid system that the C-terminal region of NS3 (residues 303–618) interacts with the N-terminal region of NS5 (residues 320–368). Further, it is shown that this same region of NS5 is also recognized by the cellular nuclear import receptor importin-β. The interaction between NS5 and importin-β and competition by NS3 with the latter for the same binding site on NS5 were confirmed by pull-down assays. The direct interaction of importin-β with NS5 has implications for the mechanism by which this normally cytoplasmic protein may be targetted to the nucleus.
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The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative time-course study using TaqMan RT–PCR
More LessFoot-and-mouth disease (FMD) is a highly contagious, economically important virus disease of cloven-hoofed animals. The objective of the present study was to examine the early pathogenesis of FMD in pigs by a quantitative time-course study. Under experimental conditions, recipient pigs were infected by contact with donor pigs affected by FMD. Every 24 h from day 1 to day 4 after exposure, two recipient pigs were selected randomly, killed and necropsied. A range of tissues were analysed by a quantitative TaqMan RT–PCR method and by titration of FMD virus on primary bovine thyroid cells. The titres of virus determined by assay in cell culture and calculated from the quantitative TaqMan data correlated strongly (r>0·9), thereby establishing the validity of the TaqMan calculations. The data indicated that the replication of virus in the lungs contributes only in small part to airborne virus excretion. Sites in the pharynx, trachea and nasal mucosa are probably more important in that regard. The sites of earliest virus infection and possibly replication in recipient pigs appeared to be in the pharynx (soft palate, tonsil and floor of pharynx). The data indicated that FMD virus replication in pigs is rapid and that the majority of virus amplification occurs in the skin. A model for the progression of infection is proposed, indicating initial spread from the pharyngeal region, through regional lymph nodes and via the blood to epithelial cells, resulting in several cycles of virus amplification and spread.
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Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site
In the life-cycle of picornaviruses, the synthesis of the viral polyprotein is initiated cap-independently at the internal ribosome entry site (IRES) far downstream from the 5′ end of the viral plus-strand RNA. The cis-acting IRES RNA elements serve as binding sites for translation initiation factors that guide the ribosomes to an internal site of the viral RNA. In this study, we show that the eukaryotic translation initiation factor eIF4G interacts directly with the IRES of foot-and-mouth disease virus (FMDV). eIF4G binds mainly to the large Y-shaped stem–loop 4 RNA structure in the 3′ region of the FMDV IRES element, whereas stem–loop 5 contributes only slightly to eIF4G binding. Two subdomains of stem–loop 4 are absolutely essential for eIF4G binding, whereas another subdomain contributes to a lesser extent to binding of eIF4G. At the functional level, the translational activity of stem–loop 4 subdomain mutants correlates with the efficiency of binding of eIF4G in the UV cross-link assay. This indicates that the interaction of eIF4G with the IRES is crucial for the initiation of FMDV translation. A model for the interaction of initiation factors with the IRES element is discussed.
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Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease
More LessThe RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.
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Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P and P–L interactions using a minigenome system
More LessThe interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV–CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161–180 amino acids and a C-terminal region of 221–241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121–160. The data suggest that N and L protein binding domains on the P protein do not overlap.
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Activity of Toscana and Rift Valley fever virus transcription complexes on heterologous templates
A transcription system for Toscana virus (TOSV) (a member of the family Bunyaviridae, genus Phlebovirus) was constructed. For in vivo expression, the TOSV transcription system uses the viral N and L proteins and an S-like RNA genome containing the chloramphenicol acetyltransferase reporter gene in the antisense orientation flanked by the viral genomic 5′- and 3′-terminal S sequences. It was found that the N and L proteins represent the minimal protein requirement for an active transcription complex. To investigate the possibility of reassortment between TOSV and Rift Valley fever virus (RVFV), the activity of their polymerase complexes was tested on their heterologous S-like RNA genomes and this showed that both virus complexes were active. Moreover, hybrid transcriptase complexes with protein components originating from the two viruses were tested on both virus templates and only the combination RVFV L + TOSV N on RVFV S-like RNA was found to be active in this assay. These results suggest that virus reassortants might be generated whenever the two viruses infect the same host.
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Infection kinetics, prostacyclin release and cytokine-mediated modulation of the mechanism of cell death during bluetongue virus infection of cultured ovine and bovine pulmonary artery and lung microvascular endothelial cells
More LessBluetongue virus (BTV) infection causes a haemorrhagic disease in sheep, whereas BTV infection typically is asymptomatic in cattle. Injury to the endothelium of small blood vessels is responsible for the manifestations of disease in BTV-infected sheep. The lungs are central to the pathogenesis of BTV infection of ruminants; thus endothelial cells (ECs) cultured from the pulmonary artery and lung microvasculature of sheep and cattle were used to investigate the basis for the disparate expression of bluetongue disease in the two species. Ovine and bovine microvascular ECs infected at low multiplicity with partially purified BTV were equally susceptible to BTV-induced cell death, yet ovine microvascular ECs had a lower incidence of infection and produced significantly less virus than did bovine microvascular ECs. Importantly, the relative proportions of apoptotic and necrotic cells were significantly different in BTV-infected EC cultures depending on the species of EC origin and the presence of inflammatory mediators in the virus inoculum. Furthermore, BTV-infected ovine lung microvascular ECs released markedly less prostacyclin than the other types of ECs. Results of these in vitro studies are consistent with the marked pulmonary oedema and microvascular thrombosis that characterize bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.
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Complete sequence characterization of the genome of the St Croix River virus, a new orbivirus isolated from cells of Ixodes scapularis
An orbivirus identified as St Croix River virus (SCRV) was isolated from cells of Ixodes scapularis ticks. Electron microscopy showed particles with typical orbivirus morphology. The SCRV genome was sequenced completely and compared to previously characterized orbivirus genomes. Significant identity scores (21–38%) were detected between proteins encoded by segments S1, S2, S4, S5, S6, S8, S9 and S10 of SCRV and those encoded by segments S1, S3, S4, S5, S6, S7, S9 and S10, respectively, of Bluetongue virus (BTV), the prototype orbivirus species. The protein encoded by SCRV genome segment 3 (VP3) is thought to be the equivalent of VP2 of BTV. Segment 7 encodes a protein homologous to non-structural protein NS2(ViP) of BTV. Analysis of VP1(Pol) (segment 1) shows that SCRV is an orbivirus, distantly related to the other sequenced species. Blot hybridizations and sequence comparisons of the conserved protein encoded by genome segment 2 (the T2 subcore shell protein) with previously identified orbiviruses confirm that SCRV is a distinct orbivirus species, unrelated to another tick-borne species, Great Island virus. The presence of SCRV in cells prepared from tick eggs suggests that transovarial transmission of SCRV may occur in ticks.
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Expression of VP5 of infectious pancreatic necrosis virus strain VR299 is initiated at the second in-frame start codon
More LessInfectious pancreatic necrosis virus (IPNV), a member of the Birnaviridae with two double-stranded RNA genome segments, encodes five proteins designated VP1 to VP5. To study the function of the 17 kDa nonstructural protein VP5 during virus replication several mutated IPNV genome segments A were constructed and included in a reverse genetics system for IPNV to obtain recombinant virus. Mutations between nt 68 and 85 or nt 94 and 103 in the noncoding region failed to yield viable virus. Only mutations located between nt 86 and 92 and downstream of nt 104 were tolerated, and viable virus could be generated. All IPNV generated showed no difference in replication compared with the wild-type IPNV, indicating that the absence of expression of VP5 did not influence virus growth in vitro. Furthermore, the results presented here indicate that initiation of translation of VP5 occurs at position 113, the second in-frame start codon.
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An immunodominant neutralization epitope on the ‘thumb’ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies
An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the ‘helix clump’) of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.
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Analysis of the molecules involved in human T-cell leukaemia virus type 1 entry by a vesicular stomatitis virus pseudotype bearing its envelope glycoproteins
Cellular entry of human T-cell leukaemia virus type 1 (HTLV-1) was studied by a quantitative assay system using vesicular stomatitis virus (VSV) pseudotypes in which a recombinant VSV (VSVΔG*) containing the gene for green fluorescent protein instead of the VSV G protein gene was complemented with viral envelope glycoproteins in trans. Most of the cell lines tested showed susceptibility to VSVΔG* complemented with either HTLV-1 envelope glycoproteins (VSVΔG*-Env) or VSV G protein (VSVΔG*-G), but not to VSVΔG* alone, indicating that cell-free HTLV-1 could infect many cell types from several species. High concentration pronase treatment of cells reduced their susceptibility to VSVΔG*-Env, while trypsin treatment, apparently, did not. Treatment of the cells with sodium periodate, heparinase, heparitinase, phospholipase A2 or phospholipase C reduced the susceptibility of cells to VSVΔG*-Env, but not to VSVΔG* complemented with measles virus (Edmonston strain) H and F proteins (VSVΔG*-EdHF), which was used as a control. Purified phosphatidylcholine also inhibited the infectivity of VSVΔG*-Env, but not VSVΔG*-G. These findings indicated that, in addition to cell surface proteins, glycosaminoglycans and phospholipids play an important role in the process of cell-free HTLV-1 entry.
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Vascular cell adhesion molecule-1 induced by human T-cell leukaemia virus type 1 Tax protein in T-cells stimulates proliferation of human T-lymphocytes
More LessHuman T-cell leukaemia/lymphotropic virus type 1 (HTLV-1), aetiologically linked to lymphoproliferative as well as inflammatory diseases, infects and activates CD4+ helper T-cells and thus alters immunoregulatory pathways. The viral regulatory Tax protein has been shown previously to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) by T-cells. To determine the functional role of this adhesion molecule, Jurkat T-cells stably expressing either Tax or both Tax and Rex (another viral regulatory protein) were used in binding and coculture assays performed with either control Jurkat cells or primary human T-lymphocytes. Evidence was provided that VCAM-1 acting in synergy with leucocyte function-associated antigen-3 promotes T-cell–T-cell interactions and increases T-cell proliferation. Interestingly, Rex was found to modulate these events. These data establish that VCAM-1 induced by Tax on T-cells thus contributes to the immunopathological process triggered by HTLV-1 infection.
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Porcine endogenous retroviruses: in vitro host range and attempts to establish small animal models
More LessUsing transgenic pigs as the source of cells or organs for xenotransplantation is associated with the risk of porcine endogenous retrovirus (PERV) transmission. Multiple proviruses are integrated into the genome of all pigs, and virus particles, some of which are able to infect human cells, are released from normal pig cells. In order to evaluate the potential risk posed by the transmission of PERVs, in vitro infection studies were performed as a basis for small animal as well as non-human primate models. In vitro infectivity was demonstrated for permanent cell lines and primary cells from a wide range of species. Productive infection was shown using reverse transcriptase (RT) assays and RT–PCR for mink, feline and human kidney cell lines, primary rhesus peripheral blood mononuclear cells (PBMCs), and baboon spleen cells and PBMCs as well as for different human lymphoid and monocyte cell lines and PBMCs. In an attempt to establish a small animal model, naive guinea pigs, non-immunosuppressed rats, rats immunosuppressed by cyclosporin-A and immunosuppressed rats treated with cobra venom factor were inoculated with PERVs produced from porcine kidney PK-15 cells, infected human 293 kidney cells and mitogen-stimulated porcine PBMCs. Animals were also inoculated with PERV-producing PK-15 and 293 cells. No antibodies against PERV and no provirus integration were observed in any of the treated animals. This suggests that productive infection of these animals did not occur in this experimental setting.
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- Animal: DNA Viruses
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Differential roles of B cells and IFN-γ-secreting CD4+ T cells in innate and adaptive immune control of genital herpes simplex virus type 2 infection in mice
More LessThe role of B, CD4+ T and CD8+ T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated. Following primary inoculation with attenuated thymidine kinase-deficient (TK−) HSV-2, B cell-deficient (μMT) mice developed a local viraemia and transient genital inflammation, suggesting a role for B cells in the innate control of local infection and inflammation. Natural antibodies are implicated in this process, as passive transfer of normal serum into μMT mice significantly reduced HSV-2 TK− shedding in the vaginal lumen, although it did not affect subsequent inflammation. Protection against lethal HSV-2 challenge was noted in HSV-2-vaccinated wild-type, CD8+ T cell-deficient and μMT mice and was characterized by strong virus-specific IFN-γ responses in vitro and delayed type hypersensitivity (DTH) responses in vivo. In contrast, CD4+ T cell-deficient (CD4−/−) mice had impaired HSV-2-specific IFN-γ production and DTH responses and succumbed rapidly to genital HSV-2 challenge. However, protective responses to HSV-2 could be induced in HSV-2-vaccinated CD4−/− mice by treatment with recombinant IFN-γ. Taken together, these results suggest that CD4+ T cells secreting IFN-γ are critical for immune protection against lethal genital HSV-2 re-infection, whereas B cells/natural antibodies have anti-viral and -inflammatory effects in the innate control of a primary infection.
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Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus
More LessMRC-5 cells are a well-characterized human diploid fibroblast cell line approved for vaccine production and favoured for the routine propagation of human cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shortening. Following delivery of the hTERT gene to MRC-5 cells using a retrovirus vector three clones were generated that (i) expressed functional telomerase activity, (ii) exhibited telomere extension and (iii) were sustained for >100 population doublings. Immortalized MRC-5-hTERT and also HCA2-hTERT human fibroblasts were both fully permissive for HCMV as determined by plaque assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of supporting the stable maintenance of an EBV-based episomal vector with efficient transgene expression when driven by the HCMV immediate early promoter. An indicator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV β-2.7 early promoter. Telomerase immortalization of human fibroblasts will thus facilitate the growth and detection of HCMV and also the generation of helper cell lines for the propagation of HCMV deletion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may therefore be suitable for additional applications in virology, cell biology, vaccine production and biotechnology.
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Evidence for interspecies transmission of oyster herpesvirus in marine bivalves
More LessSince 1991, numerous herpesvirus infections associated with high mortality have been reported around the world in various marine bivalve species. In order to determine whether these infections are due to ostreid herpesvirus-1 (OsHV1), a previously characterized pathogen of the Japanese oyster (Crassostrea gigas), PCR analysis was carried out on 30 samples of larvae collected from four bivalve species (C. gigas, Ostrea edulis, Ruditapes decussatus and Ruditapes philippinarum), most exhibiting mortality prior to collection. All samples were shown to be infected by OsHV1. Viral genomes in three samples of C. gigas and three of R. philippinarum that originated from the same hatchery were unusual in bearing a deletion of at least 2·8 kbp in an inverted repeat region. The results demonstrate that OsHV1 is capable of infecting several bivalve species, and this raises the possibility that interspecies transmission may be promoted by intensive rearing in modern hatcheries.
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Intracellular localization of the hepatitis B virus HBx protein
The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.
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A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness
There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5·9–7·4% over the complete nucleotide sequence and 4·0–5·6% in the small-S gene, and had residues Arg122, Thr127 and Lys160, characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.
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Analysis of two genomic variants of orang-utan hepadnavirus and their relationship to other primate hepatitis B-like viruses
We recently described orang-utan hepadnavirus (OuHV) (Warren et al., Journal of Virology, 73, 7860–7865, 1999). Phylogenetic analyses indicated that the various isolates of OuHV can be divided into two genomic variants. Two representatives from each genomic cluster were analysed both molecularly and phylogenetically. Their genome organization was highly similar to other hepadnaviruses of apes and humans. The complete genome sequences of the two OuHV types had an overall 5% sequence difference. Research on 25 seropositive Bornean orang-utans showed that, of the 19 animals infected with one variant, 12 originated from East Kalimantan. Phylogenetic analysis was performed using the full-length genomes of various primate hepadnaviruses. The tree topology revealed one cluster of Old World hepadnaviruses that is divided into two subclusters, one consisting of the ape viruses, and the other comprising the human genotypes A–E. These data suggest that the great apes and gibbons have been infected with a common ancestor hepadnavirus.
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Progressive multifocal leukoencephalopathy in human immunodeficiency virus type 1-infected patients: absence of correlation between JC virus neurovirulence and polymorphisms in the transcriptional control region and the major capsid protein loci
Progressive multifocal leukoencephalopathy (PML) is a rapidly fatal demyelinating disease of the central nervous system related to JC polyomavirus (JCV) replication in oligodendrocytes. PML usually occurs in immunocompromised individuals, especially in the setting of AIDS. Administration of highly active anti-retroviral therapy (HAART) may improve survival prognosis in some, but not all, patients with AIDS-related PML. This observation might be explained by the outgrowth of some JCV variants of increased fitness. To evaluate this hypothesis, two subgroups of five patients with AIDS-related PML, started on HAART after PML diagnosis, were analysed. The non-responder (NR) patients died rapidly despite HAART, while responders (R) had a positive outcome and were still alive. JCV DNA was extracted from cerebrospinal fluid biopsies and two regions of the genome were analysed, the transcriptional control region (TCR) and the major capsid protein gene (VP1). Both regions show different degrees of polymorphism and are recognized as evolving independently. Sequence analysis demonstrated that (i) extensive TCR rearrangements were present in both subgroups of patients, (ii) VP1 sequence polymorphisms could be identified in the BC loop, suggesting the absence of immune selection, and (iii) no genomic marker for JCV specific neurovirulence could be identified in the TCR and VP1 loci.
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Avian polyomavirus agnoprotein 1a is incorporated into the virus particle as a fourth structural protein, VP4
More LessAgnoproteins, encoded by the 5′-region of the late bicistronic mRNA of some polyomaviruses, are small proteins with largely unknown functions. In avian polyomavirus (APV)-infected cells, mRNAs of seven putative agnoproteins have been observed. Recently, it has been shown that agnoprotein 1a and its truncated variant agnoprotein 1b, encoded by the predominant mRNA species, are essential for APV replication. Here, the presence of agnoprotein 1a is demonstrated in the nucleus of APV-infected cells and in purified APV particles. Interaction between agnoprotein 1a and the major structural protein, VP1, was demonstrated by co-immunoprecipitation experiments using lysates of recombinant baculovirus-infected insect cells. With proteins expressed in E. coli, binding to double-stranded DNA in a sequence-unspecific manner was shown for agnoprotein 1a, whereas agnoprotein 1b failed to bind. A leucine zipper-like motif present in agnoprotein 1a is considered to be involved in DNA binding. Due to the absence of any structural or functional homologies between APV agnoprotein 1a and the agnoproteins of mammalian polyomaviruses, it is suggested that this protein should be renamed VP4, indicating its function as a fourth structural protein of APV.
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Construction and initial characterization of an infectious plasmid clone of a newly identified hamster parvovirus
More LessThe construction and characterization of a full-length infectious plasmid clone of the newly identified hamster parvovirus (HaPV) are described. Following transfection of hamster BHK cells with the infectious clone, pHaPV, the specific intracellular DNA replicative forms, RNA transcripts and viral proteins that were expected for this rodent parvovirus were generated. Infected cells were lysed and progeny virus was produced, demonstrating that pHaPV could generate a productive virus infection. The complete sequences of both hairpin termini, which had not been previously determined, were obtained. Preliminary host-range studies, which compared virus production and macromolecular synthesis in various cell lines following either HaPV infection or pHaPV transfection, demonstrated an early block of infection of HaPV in both monkey COS-1 and murine A9 cells. The availability of an HaPV infectious clone will facilitate its genetic analysis and allow the elucidation of the determinants important in host range, tissue tropism and pathogenicity of this newly identified rodent parvovirus.
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- Insect
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Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines
An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3′-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.
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- Plant
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Geographically distant isolates of the crinivirus Cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein gene
More LessThe population structure and genetic variation of Cucurbit yellow stunting disorder virus (CYSDV) isolates were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of the CYSDV coat protein gene. Analysis of 71 isolates collected from Spain, Jordan, Turkey, Lebanon, Saudi Arabia and North America showed that, from a genetic viewpoint, these isolates could be divided into two diverged subpopulations: an Eastern subpopulation composed of Saudi Arabian isolates and a Western subpopulation containing the rest of the CYSDV isolates. The genetic variation within the Western subpopulation was very small (nucleotide identity >99%) in spite of the extensive and discontinuous geographical distribution and different years of collection. We also estimated the within-isolate genetic structure and variation of three CYSDV isolates by analysing 30 clones per isolate. Our results showed that these CYSDV isolates had a quasispecies structure.
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Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system
More LessA map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus Potyvirus, family Potyviridae) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC–HC, HC–CI, VPg–VPg, NIa–NIb and CP–CP) and weak but reproducible interactions were detected for seven additional combinations (P1–CI, P3–NIb, NIaPro–NIb, VPg–NIa, VPg–NIaPro, NIaPro–NIa and NIa–NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC–HC, VPg–VPg, VPg–NIa, NIa–NIa and NIa–NIb) and weaker but reproducible interactions were detected for three additional combinations (P3–NIa, NIa–NIaPro and CP–CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg–NIa, NIa–NIaPro and NIa–NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.
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A subpopulation of RNA 1 of Cucumber mosaic virus contains 3′ termini originating from RNAs 2 or 3
More LessTobacco plants transgenic for RNA 1 of Cucumber mosaic virus and inoculated with transcript of RNAs 2 and 3 regenerated viral RNA 1 from the transgenic mRNA, and the plants became systemically infected by the reconstituted virus. cDNA fragments corresponding to the 3′ non-coding region (NCR) of viral RNA 1 were amplified, cloned and sequenced. In some clones the termini of the 3′ NCR corresponded to those of viral RNAs 2 or 3. This suggested that in some cases RNA 1 may have been regenerated during replication by a template switching mechanism between the inoculated transcript RNAs and the mRNA. However, encapsidated, recombinant RNA 1 with the 3′ NCR ends originating from RNAs 2 or 3 also was found in virus samples that had been passaged exclusively through non-transgenic plants. Thus, these chimeras occur naturally due to recombination between wild-type viral RNAs, and they are found encapsidated in low, but detectable amounts.
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Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus
More LessAlfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera Alfamovirus and Ilarvirus, respectively, of the family Bromoviridae. Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3′ termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. Cis-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV–PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
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The molecular characterization of 16 new sequence variants of Hop stunt viroid reveals the existence of invariable regions and a conserved hammerhead-like structure on the viroid molecule
More LessAt present isolates of Hop stunt viroid (HSVd) are divided into five groups: three major groups (plum-type, hop-type and citrus-type) each containing isolates from only a limited number of isolation hosts and two minor groups that were presumed to derive from recombination events between members of the main groups. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) where this viroid had not previously been described. Molecular variability comparisons considering the totality of the sequence variants characterized so far revealed that most of the variability is found in the pathogenic and variable domains of the viroid molecule whereas both the terminal right (TR) and left (TL) domains are regions of low or no variability, respectively, suggesting the existence of constraints limiting the heterogeneity of the sequence variants. Phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought. When the cruciform structure alternative to the typical rod-like conformation was considered it was observed that the upper part of this structure (hairpin I) was strictly conserved whereas in the lower part a reduced variability was found. The existence of a covariation in this lower part was notable. Interestingly, a hammerhead-like sequence was found within the TR domain of HSVd and it was strictly conserved in all the sequence variants. The evolutionary implications of the presence of this motif on the HSVd are discussed.
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