- Volume 82, Issue 8, 2001
Volume 82, Issue 8, 2001
- Review Article
-
- Animal: RNA Viruses
-
-
-
Distribution of ecotropic retrovirus receptor protein in rat brains detected by immunohistochemistry
More LessFriend murine leukaemia virus (FrMLV) FrC6 clone A8 causes spongiform degeneration in the central nervous system (CNS) of newborn but not 3-week-old rats. To assess whether expression of the ecotropic MLV receptor (CAT-1) in the CNS correlates with the pathogenicity of the A8 virus, we generated an anti-CAT-1 antibody raised against a synthetic peptide that corresponds to the carboxyl-terminal amino acid sequence of CAT-1. In the CNS of newborn and 3 to 4-week-old rats, a strong immunoreactivity against the antibody was detected in most of the endothelial cells. However, almost no expression of CAT-1 was detected in the CNS of 21-week-old rats. In newborn rats, many parenchymal cells in the brain as well as the vascular wall expressed CAT-1 antigen. These findings suggest that retrovirus receptor-bearing glial cells contribute to the neuropathogenesis of MLV, including clone A8, which induces spongiosis in rats only when inoculated into newborns.
-
-
-
-
Gender-related differences in susceptibility, early virus dissemination and immunosuppression in mice infected with Friend murine leukaemia virus variant FIS-2
More LessAn emerging amount of data indicates a correlation between gender-related factors and regulation of virus infection and supports what is known in clinical circles, that these topics are of great importance in many infectious diseases. In the present study we found that young adult NMRI male mice are more susceptible to infection by a variant of Friend murine leukaemia virus, FIS-2, than are female mice. We observed that the level of virus in serum, bone marrow and spleen was initially higher in male mice. Male mice were also more susceptible to FIS-2-induced immunosuppression. These results indicate a more efficient virus replication and dissemination in male mice. Studies with recombinant viruses between FIS-2 and the prototype Friend murine leukaemia virus revealed that FIS-2 LTR is one major factor contributing to the observed gender differences. A possible sex hormone influence on FIS-2 transcription due to the presence of a glucocorticoid response element in FIS-2 LTR is discussed.
-
-
-
Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding
More LessDifferent classes of porcine endogenous retroviruses (PERVs), which have the potential to infect humans during xenotransplantation, have been isolated from the pig genome. Because vertebrate genomes may contain numerous endogenous retrovirus sequences, the pig genome was examined for additional endogenous retroviruses, resulting in the isolation of a novel, complete endogenous retrovirus genome, designated PERV-E. The gag, pol and env genes of PERV-E are closely related to those of human endogenous retrovirus (HERV) 4-1, which belongs to the HERV-E family. Results of studies to determine the presence and copy number of PERVs demonstrated that PERV-E and PERV-A/B-like proviruses were present in all genomes tested, but that PERV-C was not found in two of the species examined, including wild boar. Multiple copies of PERVs could be found in each pig genome. Among all of the pig genomes tested, the wild boar genome had the lowest copy number of all PERVs, suggesting that the number of integrations of complete endogenous retroviruses is increased by inbreeding.
-
-
-
The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro
Recombinant measles viruses (MV) in which the authentic glycoprotein genes encoding the fusion and the haemagglutinin (H) proteins of the Edmonston (ED) vaccine strains were swapped singly or doubly for the corresponding genes of a lymphotropic MV wild-type virus (strain WTF) were used previously to investigate MV tropism in cell lines in tissue culture. When these recombinants and their parental strains, the molecular ED-based clone (ED-tag) and WTF, were used to infect cotton rats, only viruses expressing the MV WTF H protein replicated in secondary lymphatic tissues and caused significant immunosuppression. In vitro, viruses containing the ED H protein revealed a tropism for human peripheral blood lymphocytes as documented by enhanced binding and virus production, whereas those containing the WTF H protein replicated well in monocyte-derived dendritic cells (Mo-DC). This did not correlate with more efficient binding of these viruses to DC, but with an enhancement of uptake, virus spread, accumulation of viral antigens and virus production. Thus, replacement of the ED H protein with WTF H protein was sufficient to confer the DC tropism of WTF to ED-tag in vitro. This study suggests that the MV H protein plays an important role in determining cell tropism to immune cells and this may play an important role in the induction of immunosuppression in vivo.
-
-
-
Interaction between molecules of hantavirus nucleocapsid protein
More LessIntermolecular interactions of Tula hantavirus N (nucleocapsid) protein were detected in the yeast two-hybrid system, prompting further attempts to study this phenomenon. Using chemical cross-linking and immunoblotting it was shown that the N protein from purified virus and from infected cell lysates as well as recombinant protein produced in a baculovirus expression system are capable of forming dimers, trimers and multimers, thus confirming the capacity of the protein molecules to interact with each other. An ELISA format was developed in which molecules of the recombinant N protein were shown to associate non-covalently, via electrostatic interactions. Divalent cations (Ca2+, Mn2+, Mg2+, Ba2+) enhanced the process 3- to 8-fold suggesting that adequate folding of the N protein is crucial for the association. Based on these data a model for hantavirus nucleocapsid assembly is proposed, in which N molecules first trimerize around the viral RNA molecule, and then the trimers gradually assemble forming longer multimers.
-
-
-
Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases
More LessThe Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses – Nodamura virus (the prototype of the genus) and Boolarra virus – are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5′ and 3′ untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3Dpol, as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.
-
-
-
Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography
Phylogenetic analysis of the Flavivirus genus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. These phylogenies identified mosquito-borne, tick-borne and no-known-vector (NKV) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. The mosquito-borne flaviviruses revealed two distinct epidemiological groups: (i) the neurotropic viruses, often associated with encephalitic disease in humans or livestock, correlated with the Culex species vector and bird reservoirs and (ii) the non-neurotropic viruses, associated with haemorrhagic disease in humans, correlated with the Aedes species vector and primate hosts. Thus, the tree topology describing the virus–host association may reflect differences in the feeding behaviour between Aedes and Culex mosquitoes. The tick-borne viruses also formed two distinct groups: one group associated with seabirds and the other, the tick-borne encephalitis complex viruses, associated primarily with rodents. The NKV flaviviruses formed three distinct groups: one group, which was closely related to the mosquito-borne viruses, associated with bats; a second group, which was more genetically distant, also associated with bats; and a third group associated with rodents. Each epidemiological group within the phylogenies revealed distinct geographical clusters in either the Old World or the New World, which for mosquito-borne viruses may reflect an Old World origin. The correlation between epidemiology, disease correlation and biogeography begins to define the complex evolutionary relationships between the virus, vector, vertebrate host and ecological niche.
-
-
-
Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2
More LessStructure–function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2660) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein–CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396–407, 412–423 and 528–535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432–443, 436–443 and 436–447 inhibited the interaction of VLPs, but not of E2660 or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure–function analysis.
-
-
-
Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism
Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296–400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE306–314 and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE296–400 in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE306–314 synthetic peptide and this reaction was inhibited by DE296–400. Moreover, DE306–314 could block dengue virus infection of target cells in an in vitro assay. A three-dimensional model of DE revealed that the three amino acids shared by DE296–400 and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE306–314 region. Since 4E11 blocked the binding of DE296–400 to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE306–314 with target cell-displayed HSHS. Our data suggest that the DE306–314 segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE306–314 to HSHS may be antiviral reagents of therapeutic interest.
-
-
-
Establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type I interferon
More LessThe establishment of persistent infections with non-cytopathic bovine virus diarrhoea virus (ncpBVDV) is crucial for the maintenance of BVDV in cattle populations. Also, super-infection of persistently infected individuals with antigenically homologous cytopathic BVDV (cpBVDV) results in fatal mucosal disease. Persistent infection with ncpBVDV is established by infection of the foetus during the first trimester of pregnancy. It has been shown previously that foetal infection with cpBVDV does not result in persistent infection. Infection of cells in vitro has demonstrated that cpBVDV induces type I interferon (IFN), whereas ncpBVDV fails to induce IFN. In this study we demonstrate that foetal challenge with cpBVDV results in IFN production, whereas ncpBVDV does not. These findings strongly suggest that the ability of ncpBVDV to inhibit the induction of type I IFN has evolved to enable the virus to establish persistent infection in the early foetus.
-
-
-
Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon-α by human peripheral blood mononuclear cells in vitro
More LessCoxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-α synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-α. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-α by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcγRII and FcγRIII in the increased synthesis of IFN-α induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-α-producing cells in response to CVB4–IgG complexes were CD14+ cells and monocyte-enriched PBMCs. With the latter, detection of IFN-α by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-α by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcγRII and III and CAR.
-
-
-
Screening enteroviruses for β-cell tropism using foetal porcine β-cells
More LessPrimary adult human insulin-producing β-cells are susceptible to infection by prototype strains of coxsackieviruses (CV) and infection may result in impaired β-cell function and/or cell death, as shown for coxsackie B virus (CVB) types 4 and 5, or have no apparent immediate adverse effects, as shown for CVA-9. Because of the limited availability of human pancreatic β-cells, the aim of this study was to find out if foetal porcine pancreatic islets could be used as a substitute in enterovirus (EV) screening. These cells resemble human β-cells in several biological properties. CVB infection resulted in a rapid progressive decline of insulin content and reponsiveness to insulin release. The amount of virus inoculum sufficient for this destruction was small, corresponding to only 55 infectious units per pancreas. In contrast to CVBs, CVA-9 replicated poorly, and sometimes not at all, in foetal porcine β-cells. The first signs of functional impairment and cell destruction, if present at all, were seen only after 1–3 weeks of incubation. Furthermore, CVA-16, several strains of echoviruses and human parechovirus type 1 were unable to replicate in foetal porcine pancreatic β-cells. Based on these results, foetal porcine islets are somewhat more sensitive to CVB infection than adult human islets, whereas many other human EV strains do not infect porcine β-cells. Therefore, foetal porcine β-cells cannot be used for systematic screening of human EV strains and isolates for β-cell tropism, but they might provide a useful model for detailed studies on the interaction of CVBs with β-cells.
-
- Animal: DNA Viruses
-
-
-
Role of conserved residues in the activity of adenovirus preterminal protein
More LessPreterminal protein (pTP) is a component of the preinitiation complex which forms at the adenovirus origin of DNA replication and acts as the protein primer during DNA synthesis. In order to determine the role of various regions of the molecule a series of 18 mutations was introduced into conserved motifs of pTP which were predicted to be surface exposed, and the mutants expressed in insect cells using a baculovirus expression system. Their ability to initiate DNA replication was assessed and the effect the mutations have on the individual interactions which contribute to the formation of the pre-initiation complex was determined. Classes of mutants could be identified which were unable to bind DNA or interact with the adenovirus DNA polymerase, but one class of mutants retained these activities and yet failed to initiate DNA replication. These mutants therefore identify regions of pTP required for different aspects of adenovirus DNA replication.
-
-
-
-
Inhibition of transcription-regulating properties of nonstructural protein 1 (NS1) of parvovirus minute virus of mice by a dominant-negative mutant form of NS1
More LessNonstructural protein 1 (NS1) of minute virus of mice is involved in viral DNA replication, transcriptional regulation and cytotoxic action in the host cell. Viral DNA replication is dependent on the ability of NS1 to form homo-oligomers. To investigate whether oligomerization is required for NS1 transcriptional activities, a functionally impaired mutant derivative of NS1 that was able to interact with the wild-type (wt) protein and inhibit its activity in a dominant-negative manner was designed. This mutant provided evidence that transactivation of the parvoviral P38 promoter and transinhibition of a heterologous promoter by NS1 were both affected by the co-expression of the wt and the dominant-negative mutant form of NS1. These results indicate that additional functions of NS1, involved in promoter regulation, require oligomer formation.
-
-
-
Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells
More LessStructural studies have implicated Cys9, Cys104 and Cys207 of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys9, Cys104 and Cys207 continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys9→Ser change showed defects in plaque formation. Single mutants at Cys9 formed small plaques; mutants at Cys9 . Cys104, Cys9 . Cys207 and Cys9 . Cys104 . Cys207 formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys9 codon. These results further confirm the involvement of the three Vp1 cysteines in protein–protein interactions during virus assembly. Cys9 is critical for production of wild-type infectious virions, whereas Cys104 and Cys207 play secondary roles.
-
-
-
Mutational analysis of the major heparan sulfate-binding domain of herpes simplex virus type 1 glycoprotein C
More LessHeparan sulfate (HS) has been identified as a receptor molecule for numerous microbial pathogens, including herpes simplex virus type 1 (HSV-1). To further define the major HS-binding domain of the HSV-1 attachment protein, i.e. glycoprotein C (gC), virus mutants carrying alterations of either two neighbouring basic amino acid residues or a single hydrophobic amino acid residue within the N-terminal domain of the protein (residues 26–227) were constructed. In addition, a mutant lacking the Asn148 glycosylation site was included in the study. Binding of purified mutated gC proteins to isolated HS chains showed that viruses with mutations at residues Arg(129,130), Ile142, Arg(143,145), Arg(145,147), Arg(151,155) and Arg(155,160) had significantly impaired HS binding, in contrast to the other mutations, including Asn148. Impairment of the HS-binding activity of gC by these mutations had profound consequences for virus attachment and infection of cells in which amounts of HS exposed on the cell surface had been reduced. It is suggested that basic and hydrophobic residues localized at the Cys127–Cys144 loop of HSV-1 gC constitute a major HS-binding domain, with the most active amino acids situated near the C-terminal region of the two cysteines.
-
-
-
Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts
More LessIn the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38·4±4·5% were CD5+ T-lymphocytes (18·1±3·2% CD4+ 13·6±1·8% CD8+), 18·1±5·4% were B-lymphocytes, 8·5±3·9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0·974) and G2/M-phase (r=0·927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G2/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1·6 to 13·4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.
-
-
-
Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not
More LessMurine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the VH to the Vκ domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.
-
-
-
Human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) ORF50 interacts synergistically with the tat gene product in transactivating the human immunodeficiency virus type 1 LTR
More LessHuman herpesvirus-8 (HHV-8) is a lymphotropic virus associated with several AIDS-related neoplasms. Two ORFs play a critical role in the regulation of virus replication: ORF50, encoding an immediate-early transcriptional activator, and ORF57, encoding a post-transcriptional regulator. We analysed their effects on the activation of the human immunodeficiency virus type 1 (HIV-1) LTR. ORF50 interacted synergically with tat, inducing a 10-fold enhancement of HIV-1 LTR transactivation. This effect occurred both in BCBL-1 cells, latently infected with HHV-8, and in HL3T1 cells, an epithelial cell line non-permissive to HHV-8 infection. Also, ORF57 enhanced tat-induced transactivation of HIV-1 LTR, but only in BCBL-1 cells, suggesting that its action was likely mediated by the induction of other viral functions. Finally, when both ORFs were expressed, the enhancement of transactivation induced by ORF50 was partially inhibited. The findings suggest that ORF57 can modulate ORF50 activity and that ORF50 may render biologically active small amounts of tat.
-
-
-
Perforin and Fas in murine gammaherpesvirus-specific CD8+ T cell control and morbidity
The immune system uses both virus-specific T cells and B cells to control the acute and latent phases of respiratory infection with the murine gammaherpesvirus 68 (γHV-68). We sought to further define the important effector mechanisms for CD8+ T cells. First, depletion of the CD4+ T cells resulted in a failure of most animals to drive the virus into latency, although lytic virus in the lung was reduced by approximately 1000-fold from its peak. Second, the absence of either perforin or Fas alone had no impact on the ability to reduce titres of lytic virus in the lung. Further neutralization of IFN-γ in CD4-depleted P+/+, P−/− or Fas−/− mice had no effect. To define the requirements for Fas or perforin more clearly, two sets of chimeric mice were constructed differing in perforin expression by the T cells, and Fas on infected epithelial cells or lymphocytes. Animals with P−/− T cells and a Fas−/− lung failed to limit the shedding of infectious virus, regardless of whether CD4 T cells were present. In addition, we noted that having P−/− T cells in irradiated Fas+/+ hosts caused a lethal disease that was not apparent in the non-chimeric (unirradiated) P−/− (Fas+/+) mice. In another set of chimeric mice, P−/− T cells were able to limit persistent infection of B cells that expressed Fas, but not B cells that were Fas-deficient. These studies demonstrate that some degree of cytotoxicity via either perforin or Fas is essential for CD8+ T cells to control this DNA virus.
-
- Plant
-
-
-
Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus
Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the ‘BYV-type vesicles’, thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686–692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493–2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750–757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806–813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.
-
-
-
-
Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae
Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.
-
-
-
Complete nucleotide sequence and genome organization of Grapevine fleck virus
More LessThe complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3′-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5′ and 3′ ends, respectively. ORF 1 potentially encodes a 215·4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24·3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3′ end of the viral genome and encode proline-rich proteins of 31·4 kDa (p31) and 15·9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.
-
- Other Agents
-
-
-
Cellular prion protein status in sheep: tissue-specific biochemical signatures
More LessExpression of the cellular prion protein PrPC is sine qua none for the development of transmissible spongiform encephalopathy and thus for the accumulation of the illness-associated conformer PrPSc. Therefore, the tissue distribution of PrPC at the protein level in both quantitative and qualitative terms was investigated. PrPC was quantified using a two-site enzyme immunometric assay which was calibrated with purified ovine recombinant prion protein (rPrP). The most PrPC-rich tissue was the brain, followed by the lungs, skeletal muscle, heart, uterus, thymus and tongue, which contained between 20- and 50-fold less PrPC than the brain. The PrPC content of these tissues seems to be comparable between sheep. Other organs, however, showed different, but low, levels of the protein depending on the animal examined. This was also the case for tissues from the gastrointestinal tract. The tissue containing the lowest concentration of PrPC was shown to be the liver, where PrPC was found to be between 564- and 16000-fold less abundant than in the brain. PrPC was concentrated from crude cellular extracts by immunoprecipitation using several monoclonal and polyclonal anti-ovine PrP antibodies. Interestingly, it was observed that the isoform profile of PrPC was tissue-specific. The most atypical electrophoretic profile of PrPC was found in the skeletal muscle, where two polypeptides of 32 and 35 kDa were detected.
-
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)