- Volume 78, Issue 8, 1997
Volume 78, Issue 8, 1997
- Articles
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Random selection: a model for poliovirus infection of the central nervous system
More LessMixed infections occur in the natural environment, and also result from the use of mixed live vaccines. Some recipients of the trivalent oral poliovirus vaccine develop vaccine-associated paralytic poliomyelitis (VAPP). Numerous serotypes and recombinant genotypes of vaccine-derived polioviruses may be found in stool samples from such cases. To investigate the relationship between the multiplication of various genotypes at the primary replication site in the gut and the infection outcome in the central nervous system (CNS), the viruses excreted on consecutive days by two patients with VAPP were compared with the viruses isolated from the CNS. The genotypes from stools were numerous and varied with time in both cases, suggesting a multiplication of the viruses in multiple foci in the gut. Where the CNS isolated virus clearly corresponded to one of the many viruses detected in stool, this virus was unexpectedly less neurovirulent than others isolated from stool. To assess the mechanism by which viruses with different degrees of neurovirulence are selected in the CNS, transgenic mice sensitive to poliovirus infection were inoculated extraneurally with mixtures of two phenotypically different viruses at different neuropathogenic doses. The virus(es) inducing neurological disease was then isolated from the CNS. At less than 100% input neuropathogenic dose of both inoculated viruses, individual mice were affected stochastically by the virus variants from the mixture. Extrapolated to humans, this selection pattern might explain the occurrence of CNS infections with less neurotropic viruses derived from an extraneural pool containing also highly neurotropic viruses.
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Immunogenic, non-infectious polio subviral particles synthesized in Saccharomyces cerevisiae
More LessPolioviral genes coding for P1, the precursor for the structural proteins, and 3CD, the viral protease, were cloned in a Saccharomyces cerevisiae inducible expression system. N-antigenicemptycapsidscould be isolated from the yeast cell extract provided that pirodavir, a capsid-binding compound and capsid stabilizer, was added during the induction period and during purification. Purification was by immuno- affinity chromatography. The purified empty capsids had the same immunogenicity as poliovirus virions. The techniques described might be useful for the production of new non-infectious vaccines.
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Chimeric coxsackie B3 virus genomes that express hybrid coxsackievirus-poliovirus 2B proteins: functional dissection of structural domains involved in RNA replication
The 2B proteins of coxsackievirus and poliovirus (PV) share significant structural similarity and exhibit similar biochemical activities, namely inhibition of protein secretion and modification of membrane permeability. Both proteins contain two hydrophobic domains in the carboxy-terminal two-thirds of their sequence, of which one has the potential to form a cationic amphipathic α-helix. To gain more insight into the structural requirements of enterovirus protein 2B for its functioning in viral RNA replication, a chimeric cDNA approach was used. Chimeric coxsackie B3 virus (CBV3) genomes were constructed that expressed either the entire PV 2B protein or hybrid proteins in which specific segments of CBV3 2B were substituted by their corresponding PV counterparts. In vitro synthesis and processing of the chimeric polyproteins showed no abnormalities. CBV3 genomes carrying the entire PV 2B gene failed to replicate. A chimeric genomethat expressed a hybrid 2B protein consisting of the amino-terminal one-third of PV and the remainder of CBV3 yielded viable viruses. In contrast, a 2B protein consisting of the amino-terminal one-third of CBV3 and the remainder of PV failed to drive replication. These data imply that a sequence-specific interaction with another viral protein is required to drive RNA replication and suggest that the proposed sites of contact reside in the carboxy-terminal two-thirds of 2B. Hybrid genomes in which either the amphipathic α-helix or the other hydrophobic domain was replaced failed to replicate. The potential contribution of these domains to the structure and functioning of protein 2B are discussed.
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Influence of the 5′ noncoding region of hepatitis A virus strain GBM on its growth in different cell lines
More LessPrevious sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK- 4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5′ noncoding region (5′ NCR). Multiple nucleotide changes occurred in the 5′ NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5′ NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5′ NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5 NCRs from different GBM variants by replacing the 5′ NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5′ NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5′ NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5′ NCR.
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GB virus C E2 glycoprotein: expression in CHO cells, purification and characterization
A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48–56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.
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Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles
More LessWe have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunopre- cipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1·13 g/cm3 when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.
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Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants
More LessComplementary DNA encoding the ORF5 gene of a Quebec reference isolate (IAF-Klop) of porcine reproductive and respiratory syndrome virus (PRRSV) was cloned into the prokaryotic expression vectors pGEX-4T and pET21a to produce ORF5- glutathione S-transferase and ORF5-polyhistidine fusion proteins. Five hybridoma cell lines producing monoclonal antibodies (MAbs) to the 25 kDa viral envelope glycoprotein (GP5) were obtained from BALB/c mice immunized with the affinity chromatography-purified GST-ORF5 fusion protein. The polypeptide specificity of these anti-PRRSV MAbs, belonging to the IgG1 isotype, was confirmed by Western immunoblotting assays with recombinant and native viral proteins, and by radioimmuno- precipitation using [35S]methionine-labelled concentrated extracellular virus. All these MAbs showed virus-neutralizing (VN) activity, with VN titres ranging from 1:32 to 1:128. Two MAbs (IAF- 1B8 and IAF-8A8) reacted with similar titres with the modified live attenuated vaccine strain ATCC VR-2332, but all five failed to react to the prototype European strain, the Lelystad virus, in VN and indirect immunofluorescence tests. The results obtained suggest that these five anti-PRRSV MAbs are directed to serotype-specific linear neutralizing epitopes which are not affected by the absence of carbohydrate residues.
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Rinderpest virus isolates of different virulence vary in their capacity to infect bovine monocytes and macrophages
More LessThree isolates of rinderpest virus (RPV) with different in vivo virulence were able to infect and productively replicate in bovine monocytic cells. They differed in their kinetics of replication and the morphological changes induced in infected cultures. The highly virulent RPV-Saudi infected > 80% of cells within 6 days p.i. (m.o.i. = 0.1 TCID50 per cell). Under identical conditions, > 50% of cells were infected by the ‘mild’ (causes minimal mortality in vivo) isolate RPV-Egypt, whereas only 25% were infected by the avirulent RPV-RBOK. Infection by all three viruses produced infectious progeny, induced the formation of syncytia and stellate cells with long processes, and down-regulated MHC class II expression; there was no apparent effect on MHC class I nor LFA-1. RPV-Saudi was the most efficient at generating progeny virus and producing syncytia. While RPV-RBOK was the least efficient at inducing syncytia, RPV-Egypt was the least efficient for progeny virus production. In contrast, RPV-Egypt was particularly efficient at inducing stellate cell formation and down-regulating MHC class II expression. These results indicate a relationship between in vivo virulence and the characteristics of replication and induced morphological changes in monocytes/macrophages. The down-regulation of MHC class II expression would offer a means by which the virus could evade immune recognition. This would be particularly useful for the more cell-associated, but less efficient at maturing, RPV-Egypt.
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Analysis of bovine respiratory syncytial virus envelope glycoproteins in cell fusion
More LessTo compare the requirements for respiratory syncytial virus (RSV)-mediated cell fusion, the fusion (F), attachment (G) and small hydrophobic (SH) glycoproteins of bovine RSV (BRSV), ovine RSV (ORSV) and human RSV (HRSV) were expressed individually or coexpressed in either homologous or heterologous combinations in HeLa cells, using the vaccinia virus-T7 polymerase transient expression system. Cell fusion was examined by a reporter gene activation assay. Although the expression of the F protein alone or coexpression of the F and G proteins or the F and SH proteins induced cell fusion, coexpression of F, G and SH proteins induced extensive cell fusion. Coexpression of various combinations of envelope glycoproteins of BRSV, ORSV and HRSV indicated that substitution of heterologous SH protein affects the effective fusigenic properties of the BRSV F protein far more than that obtained by substituting the heterologous G protein.
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Multiplication and haemadsorbing activity of infectious salmon anaemia virus in the established Atlantic salmon cell line
More LessInfectious salmon anaemia virus (ISAV), which previously had never been isolated in any of the commercially available established fish cell lines, was successfully propagated in the continuous cell line Atlantic salmon (AS). The yield of infectious ISAV increased with the incubation time of virus- inoculated cells, demonstrated by in vivo infectivity trials in groups of Atlantic salmon. Trypsin treatment of the virus was not necessary for primary infection of AS cells with salmon-grown ISAV. The infection was non-cytopathic, but it was possible to detect virus-infected cells by a haemadsorption centre assay using Atlantic salmon erythrocytes. Pleomorphic enveloped virus particles were seen by transmission electron microscopy of infected AS cells. Elongated forms were observed, but spherical particles with diameters of 90–130 nm were commonest. Growth of ISAV was inhibited by actino- mycin D but not by 5-bromo-2-deoxyuridine treatment, which indicates that ISAV may be an aquatic orthomyxovirus.
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Kinetics of viral RNA synthesis following cell-to-cell transmission of human immunodeficiency virus type 1
More LessThe temporal appearance and levels of human immunodeficiency virus type 1 (HIV-1) tat, rev, nef, env and gag mRNA species were examined using a synchronized, one-step, cell-to-cell HIV-1 infection model involving HUT-78 cells and HIV-1 persistently infected H3B cells. Individual mRNAs were quantified by RT-PCR using RNA standards transcribed in vitro from cDNA clones. Consistent with an infection that produces high yields of virus, significant levels of env and gag mRNAs were detected in the cytoplasm of infected cells late in the infection cycle. However, at no time after infection did levels of tat, rev and nef mRNA, which encode the regulatory proteins of HIV-1, exceed their levels present in the persistently infected virus donor H3B cells. The absence of early phase induction of these mRNAs is in contrast to what is observed in cell-free HIV-1 infections or in PMA-stimulated HIV-1 chronically infected cell lines. Our results suggest that tat and rev mRNAs are already present in the cytoplasm of the persistently infected virus donor cells at levels sufficient for initiation and establishment of a highly productive infection in HIV-1 fusion-mediated infected cells. Thus, lack of sufficient Tat and Rev proteins is not likely to be the limiting factor for virus production in H3B cells, nor is increased production of these proteins likely to be the cause of the increased virus production seen following cell- to-cell transmission.
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Human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46 at levels that protect from complement-mediated destruction
Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complement- mediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.
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Human immunodeficiency virus type 1 Rev- and Tat-specific cytotoxic T lymphocyte frequencies inversely correlate with rapid progression to AIDS
Immunological correlates of AIDS-free survival after human immunodeficiency virus type 1 (HIV-1) infection are largely unknown. Cytotoxic T lymphocyte (CTL) responses are generally believed to be a major component of protective immunity against viral infections. However, the relationship between HIV-1-specific CTL responses and disease progression rate is presently unclear. Here we show in twelve HIV-1-infected individuals that detection of Rev-specific CTL precursors (CTLp) early in the asymptomatic stage, as well as detection of Rev- and Tat-specific CTLp later during follow-up, inversely correlate with rapid disease progression. No such correlation was found for detection of CTLp against Gag, RT or Nef. Further studies are required to determine whether a protective mechanism is indeed the basis of the observed correlation. The data presented are in agreement with the hypothesis that CTL against proteins that are important for early viral transcription and translation are of particular importance in protection from rapid disease progression.
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Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum
More LessTo evaluate its role in protection, immune serum was collected from four macaques which were chronically infected with live attenuated simian immunodeficiency virus (SIVmacC8) and had resisted challenge with wild-type SIVmacJ5. The immune serum was transferred to two naive cyno- molgus macaques by intraperitoneal injection (11 ml/kg). Four control macaques received an intraperitoneal injection of normal saline. One day later, all macaques were challenged with 10 MID50 of the J5M challenge stock of SIV. After challenge, all macaques became infected as determined by virus co-culture and diagnostic PCR. Virus loads in PBMC at 2 weeks post-challenge were indistinguishable between the two groups of macaques. Thus, the failure of passive immunization to transfer protection indicates that serum components alone are not sufficient to mediate the potent protection obtained using live attenuated vaccines. This is the first time that serum has been transferred from animals known to be protected against superinfection.
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Macaques infected with attenuated simian immunodeficiency virus resist superinfection with virulence-revertant virus
More LessMacaques infected with attenuated simian immunodeficiency virus (SIVmac) can resist superinfection challenge with virulent virus, showing the potential of live attenuated virus as an AIDS vaccine. Superinfection resistance does not, however, prevent the generation of virulent virus in vivo, suggesting that such virus may circumvent the resistance effect. Here, we show that three macaques already infected with the attenuated molecular clone SIVmacC8 were resistant to superinfection with virulent virus that arose in vivo following repair of a 12 bp attenuating lesion in the nef/3′ LTR. In contrast, four naive animals became infected following inoculation with blood taken from the macaque in which virulent virus arose. Loss of nef- specific cytotoxic T lymphocyte (CTL) responses followed repair of the attenuating lesion within nef in the donor animal, suggesting the possibility of escape from CTL-driven selection pressure.
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Mouse model to study the replication of primate foamy viruses
More LessA mouse model was developed to study the virus- host interaction of molecularly cloned human foamy virus (HFV) in vivo. The infectious process was analysed in two mouse strains, CBA/Ca and C57BL/ 6J, over a period of 24 weeks by PCR on DNAs from various animal tissues; virus serology was examined by immunoblotting. The infection persisted in both mouse strains and did not induce clinical symptoms. Upon infection of adult CBA/Ca mice HFV became detectable by PCR in an increasing number of organs over time. In contrast, in C57BL/6J mice, after an initial phase of dissemination, viral DNA sequences were found only in a few organs. Interestingly, the different course of infection was accompanied by differences in the antiviral immune response. In particular, C57BL/6J mice were high responders with respect to antibodies to the viral Bet protein, while CBA/Ca mice were low responders.
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Immunization with a mouse mammary tumour virus envelope protein epitope protects against tumour formation without inhibition of the virus infection
More LessBALB/c mice were immunized with the EP3 surface epitope of the mouse mammary tumour virus (MMTV) gp52 envelope protein before systemic infection with MMTV(C3H) or MMTV(SW). Analysis of the successive stages of the virus infection showed that although these mice were protected against mammary tumour formation, earlier stages of the infection were not inhibited, as reflected by the persisting superantigen-induced activation and deletion of Vβ-specificT cells. Transplacental transfer of maternal anti-EP3 immunoglobulins to newborns did not protect them from infection through the Peyer’s patches. Preincubation of the MMTVs with an anti-EP3 serum before injection, however, successfully inhibited the early stages of the infection. Results from this study show that to inhibit infection by MMTV efficiently, the virus must be neutralized before its interaction with the cell membrane, and that the affinity of the virus- membrane interaction is higher than that of the virus-antibody interaction.
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Use of the bovine leukaemia virus LTR U3 promoter for expressing antisense antiviral RNAs and competitive inhibition of viral infection in cell culture
More LessUse of viral inducible promoters which can be activated by virus-specific transactivator proteins to drive expression of antisense (as)RNA genes appears to be an attractive approach to inhibit virus infections in vivo. To this end, we have constructed an asRNA gene expressed from the bovine leukaemia virus (BLV) U3 promoter that is complementary to the R-U5 region of the BLV genome. This is the region that is most susceptible to inhibition by asRNA. With plasmid pLU, which expresses the asRNA gene under the control of the BLV U3 promoter, 75 % inhibition of virus replication was attained in CC81 cells (the molar ratio of pLU DNA over BLV proviral DNA in the transfection mixture was 5:1). Plasmid pLT, which contains only the BLV U3 promoter without any asRNA-coding region, also efficiently (up to 60%) inhibited virus replication when cotransfected with BLV proviral DNA at a ratio of 20:1. It was suggested that competition between functional and ‘empty’ viral promoters for the viral transactivator protein p38 tox could account for this inhibition. An immunoblotting assay showed that in the presence of nuclear extracts from CC81 cells exogenous BLV p38 tox specifically associates with its responsive sequence located in the BLV U3 promoter. Moreover, the additional expression of p38 tox in CC81 cells abolishes the inhibitory effect of the empty viral promoter. These observations suggest a new mechanism of BLV inhibition caused, most probably, by sequestering of the viral transactivator protein.
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Deletion mapping of functional domains in the rotavirus capsid protein VP6
More LessVP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitutethe intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.
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Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA
The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in ≈ 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in ≈ 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.
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Regulation of human papillomavirus type 16 DNA replication by E2, glucocorticoid hormone and epidermal growth factor
More LessThe E1 and E2 proteins are the only human papillomavirus (HPV) proteins required for transient replication of plasmids containing the viral origin. The E2 gene products play key roles in both viral transcription and replication. In this study we have analysed in further detail the nature of the association between E1 and E2 using a series of E2 proteins mutated in conserved regions of the N- terminal domain. These proteins were tested for their ability to activate transcription and to stimulate viral DNA replication. Several of these mutants revealed that the two functions of E2 can be separated, and that they define three widely spaced regions of the N-terminal domain which are important for DNA replication, two of which retain E1- binding activity. This suggests that E2 may have a role in viral DNA replication other than simply localizing E1 to the origin of replication. Additional important elements for regulating viral gene expression have been shown to be glucocorticoid hormones and epidermal growth factor (EGF). We show here that they may also be involved in regulating viral DNA replication. Our studies show that the addition of glucocorticoid hormone significantly stimulates viral DNA replication. In contrast, addition of EGF results in modest repression of viral DNA replication. These results have important implications for the pathogenesis of HPV infection and suggest that the relative levels of E2, glucocorticoid hormone and EGF may significantly affect the outcome of an HPV infection.
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Apoptosis of cord blood T lymphocytes by herpes simplex virus type 1
More LessWe investigated apoptosis induced by herpes simplex virus type 1 (HSV-1) in cord blood T lymphocytes by using agarose gel electrophoresis, DNA content analysis and the terminal deoxytransferase (TdT)-mediated dUTP nick end-labelling (TUNEL) method. DNA fragmentation and the hypodiploid fraction in the cell cycle were both increased in HSV-1-infected CD4 and CD8 lymphocytes stimulated with phytohaemagglutinin (PHA) compared to mock-infected lymphocytes. The percentage of cells in the S phase was decreased in HSV-1-infected CD4 and CD8 lymphocytes. HSV-1 antigen, glycoprotein D (gD) and regulatory protein ICP27 were detected in 8–18% of the hypodiploid fraction of PHA-stimulated, HSV-1-infected lymphocytes. Apoptosis was induced not only in HSV-1 antigenexpressing cells but also in cells not expressing detectable viral proteins. Addition of anti-Fas antibody, anti-Fas-ligand antibody or a mixture of both had no effect on HSV-1-induced apoptosis, indicating that the Fas-Fas-ligand pathway did not contribute to HSV-1-induced apoptosis.
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Nitric oxide production induced by herpes simplex virus type 1 does not alter the course of the infection in human monocytic cells
More LessUndifferentiated U937 cells were not susceptible to herpes simplex virus type 1 (HSV-1) infection, but after differentiation with phorbol 12-myristate 13- acetate an increase in the permissivity to the virus was observed accompanied by the production of significant levels of viral particles. High levels of nitric oxide (NO) were produced in differentiated U937 cells infected with HSV-1. This production was comparable to that observed after addition of the NO donor glycerine trinitrate. The levels of NO drastically decreased when the cells were incubated with l-monomethyl arginine (l-NMA), an inhibitor of NO synthase. Although similar levels of NO were sufficient to decrease susceptibility of U937 cells to other viruses, neither incubation with NO donors nor addition of l-NMA altered the permissiveness to HSV-1 infection. Thus, these results suggest that NO does not interfere with the replication of HSV-1 in U937 cells.
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The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle
More LessNorthern hybridizations were carried out using mRNA preparations of human cytomegalovirus (HCMV)- infected cultures and gene-specific antisense RNA probes for transcriptional analysis of the gene cluster composed of genes for DNA polymerase, glycoprotein B(gB), herpes simplex virus-infected cell protein 18.5 homologue p130 and a major DNA-binding protein corresponding to open reading frames (ORFs) UL54- UL57, respectively. Monocistronic transcripts of 5 kb and 3·7 kb were found for ORFs UL54 and UL55, respectively, and five additional high molecular mass overlapping transcripts of 14 kb, 10 kb, 10 kb, 8 kb and 6 kb were found. Mapping of 5′ ends showed that transcription was initiated at the expected distance downstream of predicted TATA elements; in the case of a UL56-specific transcript two potential initiation sites were identified. Transcription was found to terminate at the expected distance downstream of either of two prominent polyadenylation consensus motifs in the region of UL54. All transcripts were identified early in the infectious cycle, except for the UL55 (gB)-specific transcript of 3·7 kb which was not synthesized until late post-infection. However, specific immunoreactions demonstrated the presence of a gB-specific polypeptide early after infection in the absence of viral DNA synthesis. It is suggested that a bicistronic transcript of 8 kb encoded by ORFs UL55 and UL54 is involved in biosynthesis of early HCMV gB.
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Human cytomegalovirus infection results in altered Cdk2 subcellular localization
More LessHuman cytomegalovirus (HCMV) stimulates numerous cellular pathways upon infection. One of these pathways involves activation of cyclin E/Cdk2. Recent reports have demonstrated that Cdk2 is retained in the cytoplasm of cells arrested in G0 by serum deprivation, sequestered from its regulatory subunit cyclin E which is located within the nucleus. Cdk2 rapidly enters the nucleus and becomes active upon stimulation of these cells with serum growth factors. The ability of HCMV to activate cyclin E/Cdk2 in both serum-arrested cells and contact- inhibited cells suggests that HCMV infection may also result in the translocation of Cdk2 into the nucleus. In this report, we demonstrate that Cdk2 is sequestered in the cytoplasm of cells arrested in G0 by contact inhibition, as well as those arrested by serum deprivation. HCMV infection results in translocation of Cdk2 from the cytoplasm into the nucleus within 24 h of infection, both in serum- arrested and contact-inhibited cells.
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Identification of the gene coding for rhesus cytomegalovirus glycoprotein B and immunological analysis of the protein
More LessThe nucleotide sequence of the gene encoding glycoprotein B (gB) of rhesus cytomegalovirus (RhCMV) was determined and the protein characterized. The open reading frame of gB encoded a protein of 854 amino acids with 60% identity and 75 % similarity at the amino acid level to human cytomegalovirus (HCMV) gB. Cysteine residues in the extraluminal part of the protein are perfectly conserved. Out of the 16 potential N-linked glyco- sylation sites present in HCMV gB, 15 are conserved in RhCMV gB. Immunoblot analyses with antisera detected three bands of 150 kDa, 90–110 kDa and 55 kDa representing the full-length gB as well as the proteolytic cleavage products. Cross-reactivity and cross-neutralization of a number of HCMV gB-specific monoclonal antibodies with RhCMV gB indicated sharing of immunogenic epitopes between the two molecules. The RhCMV gB regions corresponding to antigenic domains AD-1, 2 and 3 of HCMV gB were immunogenic during natural RhCMV infection with the AD-1 region being the immunodominant domain. The data indicate that RhCMV might represent a useful model to investigate pathogenesis and immune surveillance of cytomegaloviruses.
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Cloning and characterization of rhesus cytomegalovirus glycoprotein B
More LessRhesus cytomegalovirus (RhCMV) infection of rhesus macaques is an important model to investigate critical issues of cytomegalovirus biology. To better understand host immunological responses to viral glycoproteins, the glycoprotein B (gB) gene of RhCMV was molecularly cloned, sequenced and characterized. Transcription analysis revealed that RhCMV gB was transcribed as a late gene. The RhCMV gB gene encoded a predicted protein of 854 amino acids that was 60% identical/75% similar to the human CMV (HCMV) gB protein. The region of HCMV gB proposed to be responsible for virus binding to host cells, fusion and cell-to-cell spread was the most highly conserved region with RhCMV gB (74% identity/85% similarity). Conserved elements included 11 of 12 cysteine residues, 14 of 16 potential N-linked glycosylation sites and cross-reactive epitopes. Metabolic labelling experiments demonstrated that RhCMV gB was proteolytically processed similarly to HCMV gB. These results are critical for investigating virus-host relationships in CMV-infected primates.
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Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the γ2 herpesvirus bovine herpesvirus-4.
Genes encoding glycoprotein gH and gL homo- logues were localized in the genome of the gamma- herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-trans- ferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo- β- N-acetylglucos- aminase-H (endoH) and endoglycosidase F-N-gly- cosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunopre- cipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31–35 kDa and diffusely migrating proteinspeciesrangingfrom45–65 kDa.Tunica- mycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31–35 kDa and the 45–65 kDa proteins were glycosylated, gp31–35 being a precursor of the 45–65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31–35 kDa (gp31–35) and 45–55 kDa (gp45–55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31–35 and gp45–55. gp110 and gp45–55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.
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Immunological control of murine gammaherpesvirus infection is independent of perforin
Perforin-mediated cytotoxic T cell killing has been suggested to be of importance in the control of noncytopathic virus infections, based on studies with lymphocytic choriomeningitis virus (LCMV). We examined the role of perforin in a mouse model of gammaherpesvirus infection using transgenic perforin-deficient mice. Previous work from this laboratory has shown that CD8 T cells are essential for the resolution of the acute lung infection and control of latently infected B cells in murine gammaherpesvirus 68 infection. The absence of perforin did not significantly affect the kinetics of either the lytic lung infection or the latent spleen infection. Lymphocytes from both perforin-deficient and control mice secreted comparable levels of IFN-γ, IL-10 and IL-6. In addition, lymphocytes from both strains had similar levels of CD3ε-dependent cytotoxic activity in the spleen, draining lymph nodes and bronchoalveolar lavage. These data indicate that the lack of perforin has little affect on the ability of mice to control an experimental gammaherpesvirus infection.
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Epstein-Barr virus-encoded LMP-1 protein upregulates the pNDCF group of nucleoskeleton-cytoskeleton-associated proteins
More LessIn a recent study, we described a group of monoclonal antibodies that identify five high molecular mass proteins which associate with intermediate filaments in the cytoplasm and accumulate in nuclear foci as well. The proteins have been designated pNDCFs, proteins associated with nuclear dots and cytoplasmic filaments. Their expression in human B cells was upregulated by Epstein-Barr virus (EBV) infection or by exposure to anti-CD40 antibodies and IL4. Phenotypically representative (type I) Burkitt’s lymphoma (BL) cell lines do not express pNDCFs or, if they do, the proteins accumulate preferentially in nuclear dots. Type III BL cell lines that have drifted to a more immunoblastic phenotype during in vitro passage and EBV-transformed lymphoblastoid cell lines (LCLs) of non-neoplastic origin express these proteins regularly at high levels. They are preferentially but not exclusively associated with vimentin filaments in the cytoplasm. Here we show that all five pNDCFs can be up- regulated by expressing the EBV-encoded membrane protein LMP1 in type I BLs. Three of them could also be upregulated in the human keratino- cyte cell line RHEK-1 by LMP1 transfection. This upregulation was paralleled by the LMP1-induced increase in vimentin expression in both cell types. One of the pNDCFs, detected by the MAb DM_4A6, accumulated in cap-like structures under the cell membrane that colocalized with membrane patches of LMP1, in addition to its localization in nuclear dots and in association with cytoplasmic vimentin filaments.
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Antibodies against vaccinia virus do not neutralize extracellular enveloped virus but prevent virus release from infected cells and comet formation
More LessVaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). EEV is important for the efficient dissemination of virus both in vivo and in vitro where it causes formation of comet-shaped virus plaques. Here, we show that EEV, in contrast to IMV, is resistant to neutralization by antibodies bound to its surface. However, antibodies against EEV can prevent comet formation in cell culture. To explain this apparent paradox, we investigated the mechanism by which antibodies inhibit comet formation and demonstrated that antibodies prevent EEV release from infected cells, and consequently comet formation, by agglutination of the virus on the cell surface. Two complementary observations allowthis conclusion: first, electron microscopy showed that infected cells incubated with medium containing anti-vaccinia virus antibodies have virus aggregates on their surface; second, culture medium from these cells contained a 4 log10 fold reduction in the physical particle/ml titre in comparison with control culture. A mechanism by which antibodies to EEV proteins provide immunological protection is thus restriction of EEV release rather than neutralization of free EEV particles.
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Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments
Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured ‘virus-like particles’ for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAgA) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAgA and a read- through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments.
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Effects of a frequent double-nucleotide basal core promoter mutation and its putative single-nucleotide precursor mutations on hepatitis B virus gene expression and replication
More LessThe basal core promoter (BCP) of hepatitis B virus (HBV) directs the transcription of both precore RNA and core RNA which code for e antigen (HBeAg) and core antigen, respectively. A double mutation in the BCP which converts nucleotide (nt) 1762 from A to T and nt 1764 from G to A is frequently observed in patients with chronic hepatitis B. We recently demonstrated that this double mutation prevented the binding of a liver-enriched factor (LEF) to the BCP, suppressed only precore RNA transcription (and hence HBeAg expression), and enhanced progeny virus production. In order to understand the mechanism for the selection of this frequent double mutation, we have extended our previous studies to further characterize LEF and to compare the effects of this double-nucleotide mutation (M1) with each single-nucleotide mutation at nt 1762 (M2) and nt 1764 (M3). Our results indicate that LEF is likely composed of a heterodimer formed between the transcription factor chicken ovalbumin upstream promoter-transcription factor (COUP-TF ) and an unidentified liver-enriched factor. Further studies reveal that both M1 and M2 prevent the binding of LEF to the BCP, suppress only precore RNA transcription, and increase the efficiency of progeny virus synthesis. In contrast, M3 retains some LEF binding activity, does not suppress HBV RNA transcription, and reduces slightly the efficiency of virus progeny synthesis. The reduced ability of M3 to replicate indicates that it has no selection advantage in itself at the level of the infected hepatocyte. In spite of its enhanced replication rate, M2 is rarely detected in HBV patients. This indicates the involvement of factors other than intracellular replication rates in the selection of these virus variants in the infected individual.
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Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus
More LessThe 5 ′ -terminal genomic region (8597 nt) of little cherry virus (LChV), a mealybug-borne closterovirus, was cloned from double-stranded RNA, and its sequence determined to complete the 16934 nt sequence of the monopartite LChV RNA genome. In the 5′ to 3′ direction, the sequence encompasses ORF 1a, encoding the conserved replicative domains of methyltransferase and helicase, and ORF 1b, encoding RNA polymerase. ORFs 1a and 1b partially overlap (in 0/ 1 configuration), and the LChV replicase is probably expressed by ribo- somal frameshifting as a fusion product with a molecular mass of 318 kDa. The N-terminal part of the ORF 1a product contains a papain-like cysteine proteinase (PCP) domain with a predicted cleavage site between Gly-619 and Ser-620. The PCP and the upstream protein domains can be aligned with the equivalent parts of the leader proteins encoded by the whitefly-transmitted lettuce infectious yellows and sweet potato sunken vein clostero- viruses. Phylogenetic reconstruction based on the aligned RNA polymerase sequences clearly suggests that the aphid-transmissible and whitefly- transmissible closteroviruses represent two distinct evolutionary lineages, with the mealybug-transmissible LChV being the most remote member of the ‘whitefly’ lineage.
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Characterization of epitopes on zucchini yellow mosaic potyvirus coat protein permits studies on the interactions between strains
C. Desbiez, A. Gal-On, B. Raccah and H. LecoqMonoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full- length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.
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Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes
More LessMicroprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a β- glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either oftwotobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for trans complementation experiments.
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Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5′ non-coding but not 3′ non-coding sequences
More LessNatural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5′ terminus of PEBV or 335 nt from the 5′ terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3′ terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5′ terminal region. In vitro translation of PEBV transcripts containing 5′ noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.
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Tubule-forming capacity of the movement proteins of alfalfa mosaic virus and brome mosaic virus
More LessThe structural phenotype of the movement proteins (MPs) of two representatives of the Bromoviridae, alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), was studied in protoplasts. Immunofluorescence microscopy showed that the MPs of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. Electron microscopy and immunogold analysis confirmed the presence of both MP and virus particles in the tubules induced by AMV and BMV. The significance of the tubule-forming properties of these viral MPs is discussed.
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Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein
More LessA series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxi- genin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.
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Evidence that DNA-A of a geminivirus associated with severe cassava mosaic disease in Uganda has arisen by interspecific recombination
Geminivirus isolates associated with the epidemic of severe cassava mosaic disease in Uganda were studied and compared with virus isolates from the part of Uganda outside the epidemic area, and with African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). Isolates of a novel type [the Uganda variant (UgV)] were detected in severely affected plants from the epidemic area, whereas those from plants outside the epidemic area were typical of ACMV. The complete nucleotide sequences of DNA-A of UgV (2799 nt) and of a Tanzanian isolate of EACMV (2801 nt) were determined and are extremely similar, except for the coat protein (CP) gene. The CP gene of UgV has three distinct regions: the 5′ 219 nt are 99 % identical to EACMV (only 79 % to ACMV); the following 459 nt are 99 % identical to ACMV (75% to EACMV); and the 3′ 93 nt are 98 % identical to EACMV (76 % to ACMV). UgV DNA-A therefore is considered to have arisen by interspecific recombination of EACMV and ACMV. Despite the hybrid nature of their CP, UgV isolates were indistinguishable from ACMV in tests with 20 monoclonal antibodies (MAbs), including seven which reacted with ACMV but not EACMV. The discontinuous epitopes detected by these seven MAbs must involve amino acids which lie in the central part of the CP (residues 74–226) and which differ in ACMV and EACMV. UgV isolates were detected in severely mosaic-affected plants from all 11 widely separated locations sampled. The probable role of recombination in geminivirus evolution in the short to medium term is discussed.
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Molecular characterization of a subgroup I geminivirus from a legume in South Africa
More LessA South African geminivirus for which we propose the name bean yellow dwarf virus (BeYDV) has been isolated from French bean (Phaseolus vulgaris cv. Bonus) showing stunting, chlorosis and leaf curl symptoms. A full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into French bean by agroinoculation, and was systemically infectious in Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum, Datura stramonium and Arabidopsis thaliana. BeYDV resembles subgroup I gemini- viruses which infect monocotyledonous plants in having a single DNA component, two non-overlapping virion-sense (V1 and V2) and two overlapping complementary-sense (C1 and C2) coding regions, and an intron within the complementary- sense coding regions that is excised to produce a C1C2 fusion protein. It is most closely related to tobacco yellow dwarf virus from Australia, the only subgroup I geminivirus previously known to infect dicotyledonous plants, although it is sufficiently dissimilar (65 % nucleotide sequence identity) to be considered a distinct virus.
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The p10 gene of Spodoptera littoralis nucleopolyhedrovirus: nucleotide sequence, transcriptional analysis and unique gene organization in the p10 locus
More LessThe p10 gene of the Spodoptera littoralis (Spli) multicapsid nucleopolyhedrovirus (MNPV) was identified. With a coding sequence of 315 nucleotides (nt), corresponding to a protein of 104 amino acids, the SpliMNPV p10 gene is the longest p10 gene known. This gene codes for a putative protein with an M r of 11130 and was found to be most closely related to the Spodoptera exigua (Se) MNPV p10 (49·4 % amino acid identity) and most distant from the Autographa californica (Ac) MNPV p10 (20·0 % amino acid identity). Characterization of the protein’s secondary structure and a comparison with other p10 protein species suggested that this p10 has an extended alpha-helical domain with high probability of forming a large coiled-coil structure. The p10 mRNA was about 1500 nt long, as de-termined by Northern blot analysis. Primer extension assay mapped three transcription start sites to a conserved baculovirus late promoter motif, TAAG. In the SpliMNPV genome, the p10 gene is not flanked by genes similar to p26 and p74, as found in SeMNPV, AcMNPV, Choristoneura fumiferana MNPV and Orgyia pseudotsugata MNPV. Instead, an open reading frame (ORF) of 945 bp is located downstream from the p10 gene and is followed by another ORF in opposite orientation, encoding the p74 protein. Upstream of the p10 sequences, an ORF of 552 bp was identified that potentially encodes a 184 amino acid protein of M r 20925, which showed 52·2 % identity with the encoded product of the SeMNPV xb187 gene.
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Volumes and issues
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