- Volume 77, Issue 9, 1996
Volume 77, Issue 9, 1996
- Review Article
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Reverse Transcriptase Jumps and Gaps
More LessIntroduction. Retroviruses are single-stranded RNA viruses of eukaryotes. Different subfamilies have been described. Avian or murine oncoviruses induce neoplasms, whereas lentiviruses, typified by human immunodeficiency virus and spumaviruses, produce persistent infections. Lentiviral infections may cause chronic disease whereas spumaviruses are apparently non-pathogenic. The retroviral life cycle is characterized by reverse transcription of their single-stranded plus (i.e. coding) RNA genome into a double-stranded DNA intermediate that integrates into the host genome. Over the last 25 years the mechanism of reverse transcription has been studied in great detail and these studies have led to the model shown in Fig. I. The result of reverse transcription is a linear double-stranded DNA molecule with a long terminal repeat (LTR) at each extremity. Synthesis of each DNA strand by the virus-encoded reverse transcriptase requires one template switch, also called a jump: the first jump is needed for synthesis of a minus DNA strand complementary to the viral RNA, the second jump for plus DNA strand synthesis.
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- Animal
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- RNA viruses
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Sodium Valproate, an Anticonvulsant Drug, Stimulates Human Immunodeficiency Virus Type 1 Replication Independently of Glutathione Levels
More LessSince modulation of the glutathione (GSH) level has been implicated in the regulation of human immuno-deficiency virus (HIV) transcription and expression, we have undertaken an analysis of the effect of sodium valproate (VPA) on HIV-1 replication. VPA, which is an anti-epileptic drug in widespread use in clinical medicine, has been shown to depress the activity of GSH reductase, an enzyme required for maintaining high cellular levels of reduced GSH. The effect of this drug on HIV-1 replication has been studied in primary infected cells, i.e. peripheral blood mononuclear cells (PBMC) and monocyte/macrophages, in the CEM-SS cell line, and in chronically infected stimulated and non-stimulated U1 cells. We have shown that VPA markedly enhanced viral replication in all infected cells tested. Virus production was induced in U1 cells by VPA treatment and the stimulatory effects of tumour necrosis factor-α, interleukin-6 and granulocyte/macrophage colony-stimulating factor were augmented. The LTR-driven gene expression in Jurkat T cells was increased. However, the elevated viral production did not correlate with the effect of VPA on the intracellular GSH level. Thus, VPA stimulated in vitro HIV-1 replication in acutely and chronically infected cells and enhanced LTR-driven gene expression. These effects were observed for concentrations that are reached in the plasma of VPA-treated patients. Therefore, although the clinical significance of these data remains to be demonstrated, these results should be considered in the choice of an anticonvulsant drug in HIV-infected individuals.
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Immunogenic Presentation of a Conserved gp41 Epitope of Human Immunodeficiency Virus Type 1 on Recombinant Surface Antigen of Hepatitis B Virus
In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones. Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus. We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg). Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles. Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans. More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed. Although HIV-1 gp160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro. These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented. Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.
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The Non-Producer Phenotype of the Human Immunodeficiency Virus Type 1 Provirus F12/HIV-1 is the Result of Multiple Genetic Variations
More LessA cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.
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Human Immunodeficiency Virus Grown in CD4-Expressing Cells is Associated with CD4
Using a CD4-capture immunoassay for gp120, several strains of human immunodeficiency virus type 1 (HIV-1) grown in CD4-expressing T lymphoblastoid cells were found to contain little CD4-reactive gp120 (0.3–1.0 ng/ml) relative to virus titre (103.2–105.0 TCID50/ml) and p24 antigen (80–1000 ng/ml). The measured CD4-reactive gp120 concentrations of HIV-1 suspensions grown in CD4-negative human neuroblastoma cells were 100- to 10000-fold greater than those of HIV-1 grown in CD4-positive lymphoblastoid cells, even though both virus suspensions contained abundant viral gp120 as shown by immunoblot assay. It was postulated that CD4 derived from host cells might be associated with virions, concealing the binding domains of gp120. CD4 association with HIV-1 virions grown in CD4-positive cells was demonstrated directly by immunoblot assay of sucrose gradient-purified virus suspensions and by specific co-sedimentation of 125I-labelled OKT4 with virions propagated in CD4-expressing cells. CD4 coating of primary HIV-1 isolates grown in peripheral blood mononuclear cells was also observed. The biological significance of CD4 coating of HIV particles remains to be determined.
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Probing the Conformation of the Human T-Lymphotropic Virus I Envelope Protein Complex with Monoclonal Antibodies
More LessWe are investigating the binding of a series of monoclonal antibodies to native and detergent-treated human T-lymphotropic virus I (HTLV-I) envelope proteins to explore their conformation. A comparison of our data with previously published findings suggests that a central neutralization domain (aa 175–200) is folded such that only short stretches are exposed at the surface of the native envelope protein complex. However, the complete domain becomes accessible after treatment with mild non-ionic detergents, suggesting that envelope subunit interaction may partially obscure this domain. We further provide immunochemical evidence that a region containing a heptad repeat in the extracellular part of the transmembrane protein is folded towards the interior of the HTLV-I envelope complex.
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Genetic Diversity of Argentine Isolates of Feline Immunodeficiency Virus
We report the nucleotide sequence and genetic diversity of part of the envelope (env) gene of four strains of feline immunodeficiency virus (FIV) isolated from Argentine domestic cats. The DNA encoding the V3 to V5 regions of the env gene of the FIV isolates were amplified by PCR, cloned and sequenced. Phylogenetic analysis revealed that the Argentine isolates did not cluster into a single group; one isolate clustered with subtype B FIV isolated in the USA and Japan, whereas the others formed a new cluster of FIV which might represent a prototype sequence for subtype E.
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Variations in Lentiviral Gene Expression in Monocyte-derived Macrophages from Naturally Infected Sheep
More LessSeventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.
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Recombinant Baculovirus-Synthesized African Horsesickness Virus (AHSV) Outer-capsid Protein VP2 Provides Protection Against Virulent AHSV Challenge
More LessAfrican horsesickness virus serotype 4 (AHSV-4) outer-capsid proteins VP2 or VP2 and VP5, prepared from single or dual recombinant baculovirus expression vectors grown in Sf9 insect cells, were administered in different amounts to horses and the neutralizing antibody responses were measured. Control and vaccinated horses were challenged with virulent AHSV-4 6 months later and monitored post challenge. The results indicated that two inoculations of extracts containing VP2 and VP5, or VP2 alone, in doses of 5 µg VP2 or more per horse, were sufficient to elicit protection against African horsesickness (AHS) disease. The recombinant VP2 protein is a potential candidate vaccine for AHS in horses.
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Phosphorylation Generates Different Forms of Rotavirus NSP5
More LessNSP5 (non-structural protein 5) is one of two proteins encoded by genome segment 11 of group A rotaviruses. In virus-infected cells NSP5 accumulates in the virosomes and is found as two polypeptides with molecular masses of 26 and 28 kDa (26K and 28K proteins). NSP5 has been previously shown to be post-translationally modified by the addition of O-linked monosaccharide residues of N-acetylglucosamine and also by phosphorylation. We have now found that, as a consequence of phosphorylation, a complex modification process gives rise to previously unidentified forms of NSP5, with molecular masses of up to 34 kDa. Treatment with phosphatases of NSP5 obtained from virus-infected cells produced a single band of 26 kDa. NSP5 could be phosphorylated in vitro by incubation of immuno-precipitates with [γ-32P]ATP, producing mainly phosphorylated products of 28 and 32–34 kDa (32–34K). In both in vivo and in vitro phosphorylated NSP5, phosphates were only found attached via serine and threonine residues. The in vitro translated NSP5 precursor polypeptide, molecular mass 25 kDa (25K), could also be phosphorylated and transformed into a 28K protein by incubation with extracts obtained from virus-infected cells, but not from non-infected cells. In addition, NSP5 labelled in vivo with [1,6-3H]glucosamine showed mainly the presence of the 26K and 28K proteins (converted to 26K by protein phosphatase treatment) suggesting that the type of protein produced is regulated according to the level of phosphorylation and/or O-glycosylation. The results also suggest that NSP5 is autophosphorylated.
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Cell Culture Isolation of Piscine Neuropathy Nodavirus from Juvenile Sea Bass, Dicentrarchus labrax
More LessA virus causing a vacuolating encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax, was isolated from brain tissue in a fish cell line (SSN-1) derived from striped snakehead, Channa striatus. The isometric, non-enveloped, 30 nm diameter virus particles were resistant to pH 2–9 and heating at 56 °C for 30 min. Infectious particles had a buoyant density of approximately 1.31 g/cm3 in CsCl. Two structural polypeptides of molecular mass 40 and 42 kDa were identified and the ssRNA consisted of two fragments of molecular mass 1.10 and 0.51 × 106 Da. From these characteristics the virus was identified as a nodavirus. Due to the broad range of susceptible fish hosts and the consistent neuropathology of the disease condition, the generic term piscine neuropathy nodavirus (PNN) is proposed for this infectious agent.
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Heterogeneity of Hepatitis C Virus Genotype 2 Variants in West Central Africa (Guinea Conakry)
An overall anti-hepatitis C virus (HCV) prevalence of 6.7% was found in a sero-epidemiological study carried out in the town of Conakry (Guinea Conakry, West Central Africa) on 1421 subjects who were either blood donors, pregnant women or in- and outpatients receiving treatment for conditions other than liver disease. Seven HCV isolates from a subsample of 73 sterile sera from this population were studied for genetic characterization and classification. The 5′NCR was analysed by the Line Probe Assay. This method assigned the isolates to genotype 2. Analysis of the 5′NCR sequences alone was unable to give a more accurate classification. Comparison of NS5b region sequences (nucleotides 7575–8196), from Guinea isolates and genotype 2 database sequences, showed evolutionary distances in the range 0.15–0.26. There was a high level of subtype heterogeneity among the genotype 2 Guinea HCV isolates. Four of the subtypes were possibly new.
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Characterizationof the NTPase Activity of Japanese Encephalitis Virus NS3 Protein
More LessJapanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (Δ1–148 and Δ1-323) forms of NS3 were engineered and expressed in E. coli as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3(Δ1–148) were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3(Δ1–323) did not. The requirements for MgCl2 and MnCl2 and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses. Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases. The substrate preference was in the order GTP > ATP ≫ UTP > CTP. Interestingly, we found that Ca2+ could not substitute for Mg2+; on the contrary, it inhibited NTPase activity. The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148–323) completely abolished the activity. Therefore, amino acids 148–323 contain a critical region required for NTPase activity.
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A Mouse-attenuated Envelope Protein Variant of Murray Valley Encephalitis Virus with Altered Fusion Activity
More LessA neutralization escape variant of Murray Valley encephalitis virus (MVE), of low neuroinvasiveness in mice and with low haemagglutination activity, had a reduced rate of replication in cultured cells during the early phase of infection compared to wild-type MVE. The variant was internalized by Vero cells at a similar rate to wild-type MVE at pH 7.4, but had reduced pH-dependent membrane fusion activity. In fusion-from-within experiments in infected mosquito (C6/36) cells, the variant had a lowered pH threshold for induction of fusion, which occurred at a reduced rate and to a lesser extent than for wild-type virus. Fusion was inhibited by monoclonal antibodies specific for envelope protein epitopes E-5 and E-8, which were implicated as determinants of fusion. These observations are discussed in relation to the regulation of MVE replication by fusion of the viral envelope with endosome membranes and, in turn, how rates of replication may affect neuroinvasion.
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A porcine CD8+ T Cell Clone with Heterotypic Specificity for Foot-and-mouth Disease Virus
Foot-and-mouth disease virus (FMDV)-specific T cell lines and clones have been obtained from a swine lymphocyte antigen (SLA) inbred miniature pig vaccinated with chemically inactivated virus. One of the clones obtained, CE3, showed a specific and heterotypic proliferation against infectious but not inactivated FMDV in the presence of syngeneic peripheral blood mononuclear cells (PBMC). Adherent cells from PBMC were sufficient to support specific activation of the clone and the proliferation was abolished when the contact between CE3 and adherent cells was prevented. Phenotypic characterization of CE3 cells revealed expression of CD2, CD25 (interleukin-2 receptor), SLA class I and SLA class II. Furthermore, the cells were highly CD8 positive but showed low expression of CD4. The expression of T cell receptor (TCR) α and β genes confirmed their T cell nature. Consistent with the CD8 phenotype, the proliferative response of CE3 was inhibited with MAbs to SLA class I and CD8. Altogether, these results indicate that CE3 is a porcine SLA class I-restricted CD8+ T cell clone, that recognizes a heterotypic FMDV antigen.
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A Study of the Cellular Immune Response to Enteroviruses in Humans: Identification of Cross-reactive T Cell Epitopes on the Structural Proteins of Enteroviruses
More LessWe have attempted to extend our understanding of the enteroviral cross-reactive T cell response in humans. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated in vitro with six different serotypes of enterovirus and 15 synthetic peptides representing conserved regions in the four structural proteins of these viruses. Upon challenge with different antigens, PBMC from donors responded specifically with proliferation and production of interferon-γ (IFN-γ). In contrast, synthesis of interleukin-4 (IL-4) or IL-10 was not detected. A T cell response to each enterovirus serotype was recorded in all individuals even though not all individuals had serum neutralizing antibody against each virus. These data confirmed previous findings that human T cells recognize enteroviral cross-reactive epitopes. Analysis of the peptide-induced IFN-γ production and proliferative response showed that the cross-reactive T cell epitopes are localized mainly in capsid protein VP2 and VP3 and to a lesser extent in VP1. Surprisingly, T cell epitopes were not identified in the most conserved structural protein of enterovirus, VP4. Immune responses were mediated by CD4+ T cells in association with MHC class II molecules. The sources of IFN-γ in response to the most immunodominant cross-reactive T cell epitopes were CD4+, CD8+ and NK cells. The two latter subsets produced IFN-γ provided CD4+ T cells were present. Since T helper 1 (Th1) cells can mediate an in vivo protective immune response against poliovirus infection in mice, our novel findings in humans merit further detailed characterization of T cells that recognize the enteroviral cross-reactive T cell epitopes.
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Screening for Membrane-Permeabilizing Mutants of the Poliovirus Protein 3AB
More LessSynthesis of the poliovirus polypeptide 3AB in bacterial cells results in an increase in membrane permeability. The alterations observed resemble those elicited by bacteriophage lytic proteins, which are presumed to cause pore formation in biological membranes. This property has been exploited in the development of an in vivo screening system that allows morphological differentiation of Escherichia coli clones expressing either wild-type 3AB or variant 3AB proteins lacking the ability to permeabilize bacteria. Expression of the wild-type 3AB gene in the presence of a chromogenic β-galactosidase substrate causes E. coli clones to stain dark blue. In contrast, bacterial mutants that synthesize 3AB proteins with alterations in the hydrophobic domain lack pore-forming activity and stain to a light blue colour, allowing differentiation from wild-type clones. This phenotypic property correlates with the rate of entry of the β-galactosidase substrate into the bacteria. The method developed here was used to screen more than 8000 E. coli clones after random PCR mutagenesis of the poliovirus 3AB gene. Our results show the existence of three different domains involved in the permeabilizing activity of 3AB protein. Twenty individual amino acid substitutions were identified in clones that showed the mutant phenotype and such bacteria displayed different reduced levels of permeability towards ONPG, hygromycin B, lysozyme and uridine. The procedure reported here may be of general interest to understand structure-function relationships in other eukaryotic proteins known to form pores.
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Temperature-Sensitive Parainfluenza Type 1 Vaccine Virus Directly Accesses the Central Nervous System by Infecting Olfactory Neurons
More LessImmunohistochemical investigation showed that intranasal inoculation of mice with a temperature-sensitive (ts) mutant of parainfluenza type 1 vaccine virus resulted in infection of some olfactory neurons as well as respiratory epithelial cells. It also disclosed the presence of viral antigens in glomeruli of the olfactory bulb but not in the secondary neurons (mitral and tufted cells). Polymerase chain reaction demonstrated the persistence of virus-specific nucleic acids in the olfactory bulb. These observations lead to the conclusion that parainfluenza virus, even with a ts phenotype, gains access to the central nervous system by infecting olfactory neurons.
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The Highly Inducible Member of the 70 kDa Family of Heat Shock Proteins Increases Canine Distemper Virus Polymerase Activity
More LessThe cellular stress response is characterized by the production of heat shock proteins (HSP) which serve important cytoprotective functions. Paradoxically, in vitro induction of the stress response promotes cytopathic effect mediated by infection with canine distemper virus (CDV). The stress-mediated increase in cytopathic effect is correlated to the formation of complexes between the viral nucleocapsid (NC) and the major inducible member of the ≈ 70 kDa family of HSP (hsp72). The objective of the present study was to document the functional significance of CDV NC-HSP interaction. Cytoplasmic NC was purified from Vero cells lytically infected with the Onderstepoort strain of CDV. Both ultrastructural variants of CDV NC interacted with both hsp72 and the constitutively expressed member of the ≈ 70 kDa family of HSP (hsp73) in a reversible and ATP-dependent manner. An effect of hsp72/73 on NC polymerase activity was demonstrated using cell-free assays derived from either Vero or HeLa cell lines. Antibody specific to hsp72 suppressed both basal and stress-enhanced polymerase activity whereas hsp73-specific antibody had no affect. Supplementation of purified hsp72/73, but not hsp73 alone, enhanced basal polymerase activity in a dosage-dependent manner. Using purified NC variants, polymerase activity was demonstrated in pre-formed hsp72/73-NC complexes but not in NC devoid of HSP. These results suggest that the stimulatory effect of the stress response upon CDV gene expression may, in part, be mediated by a reversible and direct interaction between hsp72 and the viral core particle.
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Studies on Unusual Cytoplasmic Structures which Contain Rabies Virus Envelope Proteins
More LessWe investigated unusual structures produced in BHK-21 cells infected with rabies virus (HEP-Flury strain). Sellers’ staining of the cells revealed, in addition to Negri body-like structures (inclusion bodies), production of a fuchsin-stained cytoplasmic structure (FCPS) which encircled the nucleus. The frequency of the FCPS-forming cells increased as replication progressed. The FCPS was different from the inclusion body because the former contained the viral glycoprotein (G) and matrix protein (M2) antigens, while the latter contained nucleocapsid antigens. In the early phase of infection, we observed accumulation of viral envelope antigens in a cytoplasmic structure that was considered to be expanded rough endoplasmic reticulum (rER) because of its concomitant increase in BiP content. Time-course studies suggested that the envelope antigen-containing structure, which was not stained with basic fuchsin, translocated to the perinuclear region to form the FCPS. FCPS formation was dependent on incubation temperature and was decreased at 30°C, while the development of virus-induced cytopathic effect (CPE) was delayed. When the incubation temperature was shifted up to 37°C, FCPS formation was induced again and progression of CPE was accelerated in approximate proportion to the increasing number of FCPS-positive cells. From these studies, we conclude that viral G proteins gradually accumulate in the rER with M2 protein and the expanded rER converts eventually into the FCPS, which may be closely related to accelerated host cell death.
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Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites
More LessInfluenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all three of the P proteins were determined. Results indicate that binary complexes are formed between PB1–PB2 and PB1-PA but not between PB2-PA. Therefore, we concluded that PB1 is the core subunit for assembly of the virus RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acids of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two–thirds of PA. PB2-PA binding was not detected when they were expressed in the absence of the PB1 subunit.
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- DNA viruses
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Expression of Muscovy Duck Parvovirus Capsid Proteins (VP2 and VP3) in a Baculovirus Expression System and Demonstration of Immunity Induced by the Recombinant Proteins
More LessThe gene encoding the muscovy duck parvovirus (DPV)-strain 89384 capsid proteins VP2 and VP3 was cloned in a baculovirus expression system and expressed in insect cells. The recombinant proteins were found to react with specific anti-DPV serum by Western blotting and to be located in the nucleus of insect cells (Sf9) as shown by immunofluorescence. Empty virus-like particles (VLPs) identical in size and appearance to DPV virions were observed by electron microscopy. The antigenicity and immunogenicity of the recombinant proteins were evaluated by ELISA and seroneutralization. Immunization of 3-week-old muscovy ducklings induced anti-DPV antibodies; neutralizing antibody titres were consistent with those observed in ducklings inoculated with a commercial inactivated vaccine. The way to develop these promising results is discussed.
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In vivo Replication of Hamster Polyomavirus DNA Displays Lymphotropism in Hamsters Susceptible to Lymphoma Induction
More LessUsing the whole body section hybridization technique, we monitored the organ- and age-specific pattern of replication of hamster polyomavirus (HaPV) DNA in a colony of Syrian hamsters, which are susceptible to lymphoma induction. Three phases of viral infection and replication could be distinguished: first, a phase of acute infection characterized by high levels of replication of HaPV DNA in the haemopoietic organs and the liver. This culminated 5 to 7 days post-infection (p.i.); second, at 10 days p.i., a phase of viral clearance became evident; and finally, a third phase reflected both the restriction of HaPV replication in adult hamsters and the accumulation of HaPV DNA at sites of tumour development. A remarkable conformity was observed between the tissue specificity of viral replication and the induced tumour profile: high levels of replication of HaPV DNA were restricted to cells of the haemopoietic system and lymphoid tumours were induced. As shown by in situ hybridization, the viral infection in non-haemopoietic organs was due to the dissemination of HaPV-infected blood cells.
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Extracellular Simian Virus 40 Induces an ERK/MAP Kinase-independent Signalling Pathway that Activates Primary Response Genes and Promotes Virus Entry
More LessSimian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca2+-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.
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T Cell Proliferative Responses Against Human Papillomavirus Type 16 E7 Oncoprotein are Most Prominent in Cervical Intraepithelial Neoplasia Patients with a Persistent Viral Infection
T cell proliferative responses against human papillomavirus type 16 (HPV-16) E7 protein were studied in relation to HPV status over time in 51 women originally diagnosed with abnormal cervical cytology and participating in a follow-up study. HPV-16-positive patients were grouped as having either a persistent, a cleared or a fluctuating HPV-16 infection as determined by PCR in consecutive cervical smears up until the moment of testing. Positive proliferative responses against HPV-16 E7 were found in 15/26 patients with a persistent, cleared or fluctuating HPV-16 infection (57.7%). In contrast, 0/15 patients who had been typed HPV-negative during follow-up showed positive responses (P = 0.0005). Further analysis showed positive responses to be more frequent in patients with persistent HPV-16 infections and stable or progressing cervical lesions (8/9 patients reactive, 88.9%) as compared to patients with cleared or fluctuating HPV-16 infections and stable or regressing cervical lesions (7/17, 41.1%, P = 0.04). The relatively strong T cell proliferative responses against HPV-16 E7 observed in patients with a persistent HPV-16 infection and progressive cervical lesions indicate that the effectivity of such responses cannot be predicted and apparently depends on additional factors.
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Promoter Activity of Sequences Located Upstream of the Human Papillomavirus Types 16 and 18 Late Regions
More LessThe regulation of human papillomavirus (HPV) late gene expression is difficult to analyse because the late proteins L1 and L2 are only produced in the upper layers of terminally differentiated keratinocytes. However, for the minor capsid protein L2 of HPV types 1, 6, 11 and 16, rare mRNAs or cDNAs starting 3′ of the E5 open reading frame (ORF) were previously described. In order to analyse whether the DNA region preceding the late ORFs (late upstream region, LUR) of HPV-16 and HPV-18 has promoter activity, transient transfection assays employing luciferase reporter constructs were performed. The results show that the LUR of HPV-16 and HPV-18 exhibits an orientation-dependent promoter activity in different cells. By analysing 3′-deletion mutants of the HPV-16 LUR, we identified 78 bp within the sequence between the E5 and L2 ORFs to be critical for the promoter activity. Furthermore, the analysis of a 5′-deletion mutant revealed a negative cis-regulatory element located within the E2 ORF. The HPV-16 early poly(A) signal is located downstream of the critical promoter region. Inactivation of this element by site-directed mutagenesis strongly enhanced luciferase activity. However, mutation of two potential TATA-binding protein (TBP) sites located within the critical promoter region did not abolish the activity. Altogether, these data indicate the possibility of a TATA-less promoter in the HPV-16 and HPV-18 LURs. Together with the early poly(A) signal, this potential promoter might be involved in the differentiation-dependent regulation of late gene expression.
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Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects
More LessThe cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercaptoethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the ts1 enzyme, but inhibited both ts1 and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies incombination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.
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Apoptosis: a Mechanism of Cell Killing and Lymphoid Organ Impairment During Acute African Swine Fever Virus Infection
More LessInduction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.
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DNA Sequence and Transcriptional Analysis of the UL1 to UL5 Gene Cluster of Infectious Laryngotracheitis Virus
More LessWe have cloned and sequenced the Kpnl L and M restriction fragments of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1) DNA, which are localized adjacently at the right end of the unique long region of the genome. Within the 6930 bp DNA sequence six complete open reading frames (ORFs) were identified. The predicted amino acid sequences of four of them exhibit significant homologies to the UL5 (helicase), UL4, UL3 and UL2 (uracil-DNA glycosylase) genes, which are conserved in similar arrangement in all alpha-herpesvirus genomes characterized up to now. A short ORF of 72 codons between UL3 and UL4 of ILTV is positionally homologous to the UL3.5 gene present in the genomes of different members of the Varicellovirus genus of alphaherpesviruses, but not in herpes simplex virus. The predicted ILTV protein encoded upstream of UL2 possesses limited sequence homology to the UL1 gene product of herpesviruses, the structural glycoprotein L. The presence of a N-terminal signal sequence, a conserved N-glycosylation site and two conserved cysteine residues indicates a similar function of the putative ILTV protein. Upstream of the UL1 gene of ILTV the C-terminal part of an additional ORF designated as ULO was identified, which exhibits no significant homology to known herpesvirus genes. RNA analyses indicate the expression of all detected ILTV genes including ULO.
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Bovine Interleukins 2 and 4 Expressed in Recombinant Bovine Herpesvirus 1 are Biologically Active Secreted Glycoproteins
More LessThe open reading frames encoding bovine interleukin 2 (boIL-2) and bovine interleukin 4 (boIL-4) were integrated into the unique short segment of the genome of bovine herpesvirus 1 (BHV-1) and expressed under control of the murine cytomegalovirus (MCMV) immediate-early 1 (ie1) enhancer-promoter element or the MCMV early 1 (e1) promoter. Madin-Darby bovine kidney cells infected with the recombinant viruses secreted boIL-2 or boIL-4 into the culture medium. Secretion was inhibited by the presence of brefeldin A during the infection, indicating that export from the cells was dependent on a functional Golgi apparatus. Treatment of the secreted interleukins with N-glycosidase F reduced the apparent molecular mass of recombinant BHV-1-expressed boIL-2 from 22 kDa to 16 kDa and that of boIL-4 from 20 kDa to 13 kDa, which demonstrated that both cytokines contain N-linked oligosaccharides. Digestion with neuraminidase and O-glycosidase had no detectable effect on the apparent molecular masses, suggesting that BHV-1-expressed boIL-2 and boIL-4 are not, or only slightly, O-glycosylated. In vitro experiments demonstrated the biological activity of recombinant BHV-1-expressed boIL-2 and boIL-4 by their ability to maintain the proliferation of bovine 4325 T cells and activated bovine B cells, respectively. In conclusion, we show that boIL-2 and boIL-4 are secreted from recombinant BHV-1-infected cells as biologically active glycoproteins.
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The Herpes Simplex Virus Type 1 UL8 Protein Influences the Intracellular Localization of the UL52 but not the ICP8 or POL Replication Proteins in Virus-infected Cells
We have developed a panel of 14 monoclonal antibodies (MAbs) to POL, the catalytic subunit of herpes simplex virus type 1 (HSV-1) DNA polymerase encoded by gene UL30, and one MAb to the UL52 protein, another of the seven proteins essential for replication of HSV DNA. The approximate locations of the epitopes of the polymerase-specific MAbs were identified using truncated polymerase molecules, and the antibodies were characterized in a number of immunological assays allowing eight different specificities to be recognized. These MAbs, together with a polyclonal antibody raised in rabbits against a third DNA replication protein, ICP8, were used to localize the respective proteins by immunofluorescence in cells infected with wild-type HSV-1 or the DNA replication-defective mutants ambUL8 or 2-2. In BHK cells infected with ambUL8, a mutant with an amber termination codon within the coding region of gene UL8, the UL52 protein did not enter the nucleus, although ICP8 and POL entered the nucleus in a normal fashion. The failure of the UL52 protein to be correctly transported to the nucleus was also observed in both HFL and Vero cells infected with ambUL8. In contrast, UL52 protein was transported to the nucleus in BHK cells infected with wild-type HSV-1 or with 2-2, a mutant lacking a functional UL9 protein.
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Multiple Interactions Control the Intracellular Localization of the Herpes Simplex Virus Type 1 Capsid Proteins
Herpes simplex virus type 1 (HSV-1) capsid assembly takes place in the nucleus of infected cells. However, when each of the outer capsid shell proteins, VP5, VP23 and VP26, is expressed in the absence of any other HSV-1 proteins, it does not localize to the nucleus but is distributed throughout the cell. We have previously shown that the HSV-1 capsid scaffolding protein, preVP22a, can relocate VP5 into the nucleus but does not influence the distribution of VP23. We now demonstrate that the outer capsid shell protein, VP19C, is able to relocate both VP5 and VP23 separately into the nucleus. However, nuclear localization of VP26 is only observed when VP5 is present together with either VP19C or preVP22a. Thus, pair-wise interactions involving all of the abundant capsid proteins have now been identified. Electron microscope examination of insect cells coinfected with recombinant baculoviruses expressing VP19C and VP5 reveals the presence of 70 nm diameter ‘capsid-like’ structures, suggesting that these two proteins can form the basic capsid shell.
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Replication of Herpes Simplex Virus Type 1 DNA is Inhibited in a Temperature-sensitive Mutant of BHK-21 Cells Lacking RCC1 (Regulator of Chromosome Condensation) and Virus DNA Remains Linear
More LesstsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5°C, a nonpermissive temperature. In this mutant at 39.5°C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5°C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5°C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5°C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.
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Quantification of Human Herpesvirus 6 in Immunocompetent Persons and Post-mortem Tissues from AIDS Patients by PCR
A quantitative competitive PCR assay for human herpesvirus 6 (HHV-6) was developed. Firstly, viral burden was determined in the blood of 25 healthy persons. Using 1 µg of DNA, the prevalence of HHV-6 was 36% (9/25). Eight persons had viral loads of ⩽ 32 HHV-6 genomes/µg DNA. The viral burden in the ninth individual was 1.2 × 106 HHV-6 genome copies/µg DNA, which remained constant over a period of 10 months. This demonstrates the persistence of a high HHV-6 load in the absence of apparent disease. Secondly, HHV-6 burden was determined in 100 post-mortem tissues from seven AIDS patients and three controls. For all tissues combined, there was a statistically significant higher median viral load in AIDS patients (56 copies/µg DNA, range 0–43 321) compared to controls (10 copies/µg DNA, range 0–423) (P = 0.04). The precision and reproducibility of this assay will allow hypotheses concerning the pathogenic potential of HHV-6 to be tested quantitatively.
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Glycoprotein gH of Pseudorabies Virus is Essential for Penetration and Propagation in Cell Culture and in the Nervous System of Mice
Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL. For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a β-galactosidase expression cassette into a partially deleted gH gene. The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes. After intranasal inoculation into mice, the LD50 of complemented gH− PrV was more than four orders of magnitude higher than that of wild-type PrV. Infection of the respiratory epithelium was much less efficient with complemented gH− PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread. Complemented gH− PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed. In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice. This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.
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Human Cytomegalovirus Inhibits Peptide Translocation into the Endoplasmic Reticulum for MHC Class I Assembly
More LessHuman cytomegalovirus (HCMV) genes expressed in the early phase of infection mediate the destabilization of nascent major histocompatibility complex (MHC) class I molecules in infected cells and thus prevent antigen presentation to CD8+ T lymphocytes. We report that HCMV genes interfere with the MHC class I pathway of antigen presentation by at least two mechanisms. Firstly, permissive infection of fibroblasts is characterized by a continuous decline in the capacity to translocate peptides from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Inactivation of peptide transport is operative despite augmented TAP expression during HCMV infection. Secondly, TAP molecules fail to associate with MHC class I heavy chains indicating that HCMV early gene expression also interferes with MHC class I maturation. A temperature-sensitive mutant of HCMV, ts9, which lacks 15 kb of DNA encoding the genes US1–US15 of HCMV, had lost the capacity to interfere with MHC class I assembly and to inhibit the peptide translocation function of TAP. One of the genes deleted in ts9, US11, which was reported to downregulate the expression of MHC class I molecules, does not affect peptide transport by TAP. Therefore, we conclude that HCMV encodes at least two early gene functions that interfere with the MHC class I antigen presentation pathway.
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Structural Domains Involved in Human Cytomegalovirus Glycoprotein B-Mediated Cell-cell Fusion
More LessA novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing authentic or mutagenized human cytomegalovirus glycoprotein B (HCMV gB; gpUL55). Compared to transfectants expressing authentic HCMV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hd1; aa 714–747) or with deletions of specific segments in the cytoplasmic tail (aa 811–825 and 871–906) exhibited significantly reduced heterologous fusogenicity. HCMV gB-specific monoclonal antibodies (MAbs) as well as MAb against cellular annexin II prevented fusion of the transfectant expressing authentic gB. Comparable surface exposure of HCMV gB or its derivatives was demonstrated in all transfectants by FACS analysis. Our observations are compatible with the notion that indigenous fusion activity of HCMV gB depends on the extracellular hd1 domain and on the conformation of the cytoplasmic tail.
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- Insect
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In vitro Characterization of a Trichoplusia ni Single Nucleocapsid Nuclear Polyhedrosis Virus
More LessA Trichoplusia ni single nucleocapsid nuclear poly-hedrosis virus (TnSNPV) isolate was cloned and its replication studied in the BTI-Tn-5B1-4 insect cell line. The BTI-Tn-5B1-4 cells were highly susceptible to TnSNPV infection, with 99% of the cells containing viable polyhedra by 30 h post-inoculation. Viral DNA synthesis was detected by 9 h post-infection (p.i.). Infectious budded virus (BV) was first detected at 13 h p.i. and reached an average maximum titre of 3.875 × 106 p.f.u./ml 27 h p.i. A total of 25 BV structural proteins having apparent molecular masses ranging from 27.5 kDa to 86 kDa were identified. Using [35S]methionine pulse-labelling, 19 virus-induced proteins with molecular masses ranging from 27 kDa to 106 kDa were detected from 4 to 28 h p.i. Host cell protein synthesis continued throughout virus replication, although at gradually decreasing rates. Thirty-two structural proteins of occlusion-derived virus ranging in apparent molecular masses from 11 kDa to 98 kDa were identified using silver staining procedures. Digestion of viral DNA with the restriction endonucleases EcoRI, HindIII and BamHI generated 31, 26 and 12 fragments, respectively. Estimates for the molecular mass of the TnSNPV genome ranged from 115.5 to 119.2 kbp. In bioassays performed with neonate T. ni larvae, the mean LD50s for the TnSNPV and Autographa californica MNPV were 1.5 (±0.3) and 11.0 (±4.0) polyhedra per larva, respectively.
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Sequence and Transcriptional Analysis of the Ubiquitin Gene Cluster in the Genome of Spodoptera exigua Nucleopolyhedrovirus
More LessThe nucleotide sequence of a 1200 bp DNA fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was determined. This sequence contained a cluster of two open reading frames (ORFs), one coding for a viral ubiquitin (v-ubi) and another with homology to orf2 of Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. The v-ubi ORF is 240 nucleotides (nt) long, potentially encoding a protein of 80 amino acids with a predicted molecular mass of 9.4 kDa. The amino acid sequence of the v-ubi gene in SeMNPV has 75% and 81.6% identity with the v-ubi gene of AcMNPV and OpMNPV and approximately 84% with cellular ubiquitins. Northern blot analysis revealed three major small transcripts late in infection, of about 690, 550 and 400 nt long. Primer extension analysis showed that transcription started from within two consensus late promoter elements (TAAG), located at positions -6 and -30. The start site at position -4/-5 precedes the shortest leader eported to date for a baculovirus gene. The other ORF, xb187, was identified in the opposite orientation immediately upstream of the v-ubi gene. This ORF potentially encodes a 22 kDa protein with unknown function and about 60% amino acid similarity to the products of the orf2 genes of AcMNPV and OpMNPV. The SeMNPV xb187 ORF is transcribed late in infection via two transcripts, 1.2 kb and 770 nt long. The v-ubi-xb187 gene cluster is located at map unit (m.u.) 89 on the genome of SeMNPV. This is different from the position of an identical cluster in the AcMNPV and OpMNPV genomes, located at relative m.u. 20.
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Ichnovirus Infection of an Established Gypsy Moth Cell Line
More LessIn the present study, a lepidopteran cell line (Ld-652Y, from the gypsy moth, Lymantria dispar) exposed to Hyposoter fugitivus polydnavirus (HfPV) was found to display a variety of cytopathic effects. These included a transient inhibition of cell proliferation, rounding up, aggregation and apoptosis. In addition, unusual paracrystalline structures appeared within the lumen of the rough endoplasmic reticulum; similar structures were observed in the spherulocytes of parasitized Malacosoma disstria. Following Coomassie Blue staining, two new cell-associated polypeptides were detected; one of these, an 8 kDa polypeptide, could also be observed following exposure of Ld-652Y cells to media taken from infected cultures or to cell-free haemolymph from parasitized M. disstria. After a period of 2–4 weeks, the L. dispar cell cultures were observed to largely recover from the effects of exposure to virus, and resumed proliferation; ‘transformed’ cell populations tended to form aggregates, and adhered less tightly to the substrate. Viral DNA was stably maintained in all recovered cell lines, possibly in chromosomally integrated form.
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- Plant
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Comparison of two DNA Viruses Infecting the Marine Brown Algae Ectocarpus Siliculosus and E. Fasciculatus
More LessThe marine brown algal genus Ectocarpus contains two species, E. siliculosus and E. fasciculatus. Field populations of both species include plants with infection symptoms caused by DNA viruses. We have established clonal cultures from infected and normal host plants and investigated the properties of the endogenous viruses. Both host species contain virus particles with a hexagonal cross-section and a diameter of ca. 150 nm. The genomes of both virus types consist of double-stranded DNA, approximately 320 kb in size. Restriction digestion with Sfil revealed differences between the two virus genomes. However, PCR experiments suggest that at least one gene, which encodes a major capsid protein, is quite similar in both virus species. In cross-infection experiments the E. siliculosus virus did not initiate an infection cycle in E. fasciculatus. In contrast, the E. fasciculatus virus infected E. siliculosus zoospores. The resulting plants showed aberrant symptoms and produced virus particles which were not infectious. We conclude that the two Ectocarpus species are hosts for different, but closely related viruses.
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Complete Nucleotide Sequence and Organization of the RNA Genome of Groundnut Rosette Umbravirus
More LessComplementary DNA clones representing the entire genome of groundnut rosette umbravirus (GRV) were obtained and sequenced. GRV RNA comprises 4019 nucleotides and contains four large open reading frames (ORFs). The second ORF from the 5′ end includes sequences that encode motifs characteristic of viral RNA-dependent RNA polymerases and is probably expressed by a -1 frameshift mechanism as a fusion protein with the product of the 5′-most ORF. The other two ORFs are almost completely overlapping in different reading frames, and are probably expressed from subgenomic RNA. One of the putative products has significant sequence similarity with viral movement proteins. None of the putative proteins encoded by GRV RNA seems to be a structural protein. In genome organization and in the amino acid sequences of its potential products, the RNA of GRV is similar tot hat of carrot mottle mimic umbravirus, and to the umbravirus-like RNA-2 of pea enation mosaic virus.
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The Complete Nucleotide Sequence and Genome Organization of the M RNA Segment of Peanut Bud Necrosis Tospovirus and Comparison with other Tospoviruses
The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3′ large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58–59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21–23% identity and 44–47% similarity with those of other members of the genus Bunyavirus. The 5′ small ORF (924 nucleotides in the virus-sense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41–43% identity and 60–63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.
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Addition of Nucleotides Similar to Deleted CAA Repeats in the 5′ Non-coding Region of Tomato Mosaic Virus RNA Following Propagation
More LessPreviously we made a series of deletion mutants in the 5′ non-coding region of tomato mosaic tobamo-virus (ToMV) RNA and checked their ability to replicate in tobacco protoplasts. Long deletions in this region caused the virus to lose the ability to replicate. Several mutants with deletions of about 10 nucleotides (short deletion mutants; SDM) retained the ability to replicate. In this study, we inoculated SDMs onto systemic host plants and observed their symptoms. One mutant (19/32) caused severe mosaic symptoms on some tobacco plants (Nicotiana tabacum cv. Samsun) but no symptoms on others. Virus accumulation in 19/32-inoculated plants paralleled the severity of symptoms. Four CAA repeat sequences were deleted in 19/32. Progeny 19/32 from plants showing severe systemic mosaic symptoms had acquired additional nucleotides in this region. We conclude that the CAA repeat sequence is related to the fitness of the virus population to replicate in whole plants rather than to translation of ToMV replicase genes.
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Unusual Sequence Relationships Between Two Isolates of Citrus Tristeza Virus
More LessThe complete, 19226 nt sequence of the RNA genome from VT, a seedling yellows strain of citrus tristeza virus (CTV), was determined and found to have a genome organization identical with that of the previously determined CTV-T36 isolate, except that ORF 1 of CTV-VT was 70 nt shorter due to two widely separated 18 nt deletions. Sequence comparison of CTV-VT and CTV-T36 revealed approximately 89% identity throughout the ten 3′ ORFs, but only 60–70% identity throughout ORF 1. The 5′ nontranslated regions were only 60% identical whereas the 3′ nontranslated regions were 97% identical. The transition between regions of similarity and deviation was gradual, suggesting that the sequence similarities and differences compared to CTV-T36 were unlikely to have arisen from a recent recombination event between a close T36 relative and a distantly related CTV isolate. This is the first attempt to compare in detail the variation between the genomes of two strains of a member of the closterovirus group. The observed deviation between the large RNA genomes of the two CTV strains is greater than that among different viruses of most other groups, raising the question of how to define the taxonomy of these viruses.
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The 24 kDa Proteinases of Comoviruses are Virus-specific in cis as Well as in trans
More LessTo investigate the specificity of comoviral 24 kDa (‘24K’) proteinases, a full-length cDNA copy of red clover mottle virus (RCMV) RNA 1 has been cloned downstream of a T7 promoter. Translation in rabbit reticulocyte lysates of in vitro transcripts from this clone resulted in the synthesis of a 200K protein which was processed in a manner similar to that of the equivalent protein from cowpea mosaic virus (CPMV). Full-length cDNA clones of the RNA 1 molecules of RCMV and CPMV were used to create hybrid RNA 1 molecules. RNA transcribed in vitro from these hybrids was translated in vitro and the ability of the 24K proteinase from one comovirus to cleave the 32K/170K processing site from the other assessed. The results of the experiments show that the 24K proteinases are virus-specific in cis.
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- Other Agents
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Electrophoretic Analysis of Nucleic Acids Isolated from Scrapie-infected Hamster Brain
More LessThe purpose of this study was to investigate previous reports of a scrapie-specific 1.2 kb single-stranded DNA observed in alkaline agarose electrophoresis gels. Protocols were developed to be as consistent as possible with those used previously. Partial subcellular fractionation was applied to the brains of hamsters clinically affected by the 263K strain of scrapie. Nucleic acids were then isolated, and compared electrophoretically to nucleic acids isolated from equivalent fractions, made from the brains of hamsters inoculated with normal brain. Several modifications to the protocols were suggested. Results obtained by using these modifications were compared to results obtained using the original protocols. Scrapie-specific DNA was not observed in a total of eleven consecutive experiments. Thus, these results do not support the hypothesis that the agents of transmissible spongiform encephalopathy contain a single-stranded 1.2 kb DNA species as previously described.
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