- Volume 78, Issue 12, 1997
Volume 78, Issue 12, 1997
- Articles
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Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene
Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.
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Binding and fusion of Autographa californica nucleopolyhedrovirus to cultured insect cells
More LessBinding of baculoviruses to insect cells and fusion of the virus envelope to cell membranes are early events suggested to be affected by baculovirus enhancins. The binding of Autographa californica nucleopolyhedrovirus (AcMNPV) to the Spodoptera frugiperda cell line Sf21 and the fusion of the virus envelope to cell membranes were characterized. Virus binding assays demonstrated that AcMNPV budded virus (BV) bound to specific binding sites on Sf21 cells with an avidity of 2·3 × 1010M 1. The cells displayed 3·1 × 103 specific binding sites per cell in a confluent monolayer. In addition, the effects of pH, buffer composition and cation concentration on the binding were examined. Using a fluorescent probe (R18) and fluorescence microscopy, the fusion of AcMNPV BV envelope to the cell membrane was directly visualized in living cells. It has been reported that Trichoplusia ni nucleopolyhedrovirus enters Sf21 cells by membrane fusion at the cell surface; however, the present studies confirmed the well established concept that adsorptive endo-cytosis is the major entry pathway for baculovirus BV infection. Membrane fusion kinetics and fluorescence microscopy demonstrated and verified that the envelope-cell membrane fusion was triggered by acidification. The effect of a T. ni granulovirus enhancin on virus binding and membrane fusion was examined, and no increase in activity was observed.
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Analysis of p74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo
More LessIn nature, nuclear polyhedrosis viruses (NPV) are transmitted when susceptible insect larvae ingest viral occlusion bodies (OB). These dissociate in the alkaline environment of the midgut and release encapsulated virions (PDV) which bind to midgut epithelial cells and initiate an infection. A previous study showed that expression of the Autographa californica NPV (AcMNPV) p74 gene during replication is essential for the production of infectious OB. A set of p74 deletion and overexpression recombinants was used for the production and screening of monoclonal antibodies, and for an investigation of gross cytopathology and localization of p74. No differences in virus structure or morphogenesis were observed in infected cells when the p74 gene of AcMNPV was deleted, even though the infectivity of OB harvested from the cells was abolished when they were fed to Trichoplusia ni larvae. Mutant OB released virus particles and degraded insect peritrophic membrane as in infections with wild-type virus; in addition, virions purified from mutant OB were infectious when injected into the haemocoel of T. ni larvae. Western blot analysis confirmed that p74 was associated with the PDV and could not be detected in the budded form virion phenotype. The polypeptide was readily degraded by treatment of purified PDV with proteinase K, in the presence and absence of detergent, and could be extracted from PDV by a non-ionic detergent treatment. The data are consistent with p74 being a structural polypeptide of the PDV phenotype, most probably as a component associated with the outside surface of the virion envelope. Its presence is shown to be essential for primary infection of midgut cells of insect larvae.
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Characterization of a putative Spodoptera exigua multicapsid nucleopolyhedrovirus helicase gene
More LessPutative baculovirus helicases have been implicated as playing an important role in viral DNA replication and host specificity. The Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) helicase is therefore of interest since the virus only infects the beet army worm. Sequence analysis of the SeMNPV lef5-p39 (mu 46·5–55·1) region, which is collinear with the 39K-lef5 area in Autographa californica MNPV (AcMNPV), revealed an open reading frame (ORF) of 3666 bp potentially encoding a protein with a molecular mass of 143 kDa. This protein had considerable amino acid sequence similarity (58%) to AcMNPV p143, including seven conserved motifs characteristic of helicases. In cultured insect cells, this SeMNPV ORF is expressed from 4 to 12 h postinfection and its major transcript of 4 kb starts 11 to 12 nt upstream of the putative translational initiation site (ATG). To study their possible role in the specificity of baculovirus DNA replication, the putative AcMNPV and SeMNPV helicase genes were tested for their ability to replicate homologous regions (hrs; putative origins of DNA replication) in a transient DNA replication assay in insect cells. All viral cis- and trans-acting factors were provided as plasmids using either Achr2 or Sehr1 as the DNA replication origin. SeMNPV p143 could not substitute for AcMNPV p143 in the transient assays supplemented with either hr. Similar results were obtained when the SeMNPV and AcMNPV ie1 genes were exchanged. None of the essential AcMNPV trans-acting factors could be complemented by SeMNPV infections to support DNA replication of hrs. These data suggest a specific interaction between baculovirus DNA replication factors to form the replisome and/or between the replisome and the origin of DNA replication.
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Mapping of the Heliothis armigera entomopoxvirus (HaEPV) genome, and analysis of genes encoding the HaEPV spheroidin and nucleoside triphosphate phosphohydrolase I proteins
More LessThe genome of Heliothis armigera entomopoxvirus (HaEPV) has been mapped with four restriction endonuclease enzymes (BamHI, HindIII, PstI and XhoI), and its length estimated at 233 kbp. An EcoRI- generated HaEPV genomic fragment hybridized to all fragments identified as genomic termini, providing the first experimental evidence for the presence of terminal repeat elements in an EPV genome. The HaEPV spheroidin and nucleoside triphosphate phosphohydrolase I (NPHI) genes have been cloned and sequenced, and their deduced products shown to possess high levels of identity with homologues from other Genus B entomopoxviruses (EPVs). The genomic locations of these and other HaEPV genes and open reading frames have been determined; comparison of their locations with those of homologues in the Amsacta moorei EPV genome largely supports an hypothesis that the Genus B EPVs share a conserved genomic organization which differs from that of chordopoxviruses. It is proposed that genes of EPVs can be assigned to five actual or predicted homology-based groups, a categorization which is useful for directing and interpreting investigations of EPV gene functions and relationships.
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Excision of the polydnavirus chromosomal integrated EP1 sequence of the parasitoid wasp Cotesia congregata (Braconidae, Microgastinae) at potential recombinase binding sites
More LessCotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp Cotesia congregata. To determine the molecular mechanisms for the vertical transmission of CcPDV in the wasps, we analysed the different forms of the virus sequences containing the gene encoding the early parasitism-specific protein 1 (EP1). By a detailed molecular analysis, we demonstrated that the EP1 sequences are present in wasp DNA in two forms: a circular form as seen in the virus particles and a form integrated into the wasp genome. Moreover, we showed that the integrated form of the EP1 sequences is able to excise in the ovary cells. A fragment corresponding to an EP1 ‘empty locus’(without the viral sequence) was PCR-amplified from ovarian DNA. Comparison of the sequences isolated from the EP1 circle, the integrated form and the empty locus revealed that the extremities of the EP1 genomic sequences constitute a direct repeat. Strikingly, these sequences contain a potential binding site for a recombinase of the Hin family located in close vicinity to the position where the DNA strand exchange occurs. Thus, the data bear upon the possibility that the bracovirus circles are excised via a mechanism related to the Hin mediated Conservative Specific-Specific Recombination (CSSR) of prokaryotes.
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Hypothesis on particle structure and assembly of rice dwarf phytoreovirus: interactions among multiple structural proteins
More LessTo study the morphogenesis and packaging of rice dwarf phytoreovirus (RDV), the interactions among multiple structural proteins were analysed using both the yeast two-hybrid system and far-Western blotting analysis. The following protein-protein interactions were observed. P3 (major core protein) bound to itself as well as to P7 (nucleic acid-binding protein) and P8 (major outer capsid protein). P7 bound to P1 (RNA-dependent RNA polymerase) and P8, in addition to P3. Based on these findings, we hypothesize that the core shell structure is based on P3-P3 interactions and that P7 has the ability to bind to multiple structural proteins as well as to genomic RNAs during viral particle assembly. Based on the observed protein-protein interactions and on computer-aided analysis of the numbers of structural proteins per particle, possible RDV assembly events are proposed.
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Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y
More LessA full-length cDNA clone of the potato virus Y (PVY) genome was obtained after cloning difficulties in Escherichia coli were overcome. These difficulties were mainly due to the expression of the CI gene from upstream prokaryotic promoter-like elements within the PVY genome. To overcome this problem, PVY cDNA was maintained in two subclones which were ligated before infection. A plasmid in which these two fragments were contained could be propagated in some E. coli strains but was unstable and yielded only low levels of plasmid DNA. Replacement of the 35S promoter by the SP6 promoter slightly increased the stability of the plasmid and its RNAtranscripts were infectious when capped with m7G(5′)ppp(5′)G. Using two inoculation methods (mechanical or biolistic) the cDNA and its RNA transcript proved infectious on three Nicotiana species and on Solanum tuberosum.
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35S promoter-driven cDNAs of barley mild mosaic virus RNA1 and RNA2 are infectious on barley plants
More LessFull-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically infected leaves was demonstrated by RT-PCR and Western blot analyses, respectively. Virions were detected by immunogold labelling, demonstrating that the RNAs are encapsidated. This is the first report of the 35S promoter used in successfully infecting a monocot plant host with cDNA from a strictly monocot plant RNA virus.
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Variability of geographically distinct isolates of maize rayado fino virus in Latin America
More LessWe have examined the molecular epidemiology of the leafhopper-borne maize rayado fino virus (MRFV) in Latin America. The coat protein gene and 3′ non-translated region of 14 isolates of MRFV collected from Latin America and the United States were sequenced and phylogenetic relationships examined. The nucleotide sequence revealed remarkable conservation, with a sequence similarity of 88–99 %. Phylogenetic analysis of sequence data obtained from a 633 bp fragment showed that MRFV has diverged into three main clusters, i.e. the geographically distinct northern and southern isolates and the Colombian isolates. Significant differences between the isolates collected from Colombia, previously named maize rayado colombiana virus, based upon differences in symptomatology and serological relationships to MRFV, and the other MRFV isolates, provides additional evidence supporting its designation as a unique strain of MRFV.
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Beet soil-borne virus RNA 1: genetic analysis enabled by a starting sequence generated with primers to highly conserved helicase-encoding domains
More LessThe complete sequence of the 5834 nucleotides of RNA 1 of beet soil-borne furovirus (BSBV, Ahlum isolate) was determined using a PCR product obtained with primers to highly conserved coding regions for helicase-like proteins in RNA 1 of furo-, hordei- and tobraviruses as a starting sequence. Unknown parts of the sequence upstream and downstream of this starting sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 1 contains one large ORF for a readthrough protein with a molecular mass of 204 kDa (204K protein) which is interrupted internally by a UAA stop codon terminating the coding region for a protein of 145 kDa (145K protein). The N- and C-terminal parts of the 145K protein and the readthrough domain of the 204K protein contain methyl- transferase, helicase and RNA-dependent RNA polymerase motifs, respectively. Unlike other furo- and tobraviruses BSBV contains no further genes on its RNA 1.
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Isolation and characterization of tubular structures of cowpea mosaic virus
More LessTubular structures involved in the cell-to-cell movement of cowpea mosaic virus (CPMV) were partially purified from infected cowpea protoplasts to identify the structural components. A relatively pure fraction could be obtained by differential centrifugation and this was analysed by PAGE and immunoblotting. Besides the movement protein (MP) and capsid proteins (CP) of CPMV, no other major infection-specific proteins could be detected, suggesting that host proteins are not a major structural component of the movement tubule.
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Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus
More LessAlfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) are tripartite positive-strand RNA plant viruses that encode functionally similar translation products. Although the two viruses are phylogenetically closely related, they infect a very different range of natural hosts. The coat protein (CP) gene, the movement protein (MP) gene or both genes in AMV RNA 3 were replaced by the corresponding genes of PNRSV. The chimeric viruses were tested for heterologous encapsidation, replication in protoplasts from plants transformed with AMV replicase genes P1 and P2 (P12 plants) and for cell-to-cell transport in P12 plants. The chimeric viruses exhibited basic competence for encapsidation and replication in P12 protoplasts and for a low level of cell-to-cell movement in P12 plants. The potential involvement of the MP gene in determining host specificity in ilarviruses is discussed.
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Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates
More LessHop stunt viroid (HSVd) is able to infect a number of herbaceous and woody hosts, such as grapevine, Citrus or Prunus plants. Previous phylogenetic analyses have suggested the existence of three major groups of HSVd isolates (plum-type, hop-type and citrus-type). The fact that these groups often contain isolates from only a limited number of isolation hosts prompted the suggestion that group-discriminating sequence variations could, in fact, represent host-specific sequence determinants which may facilitate or be required for replication in a given host. In an effort to further understand the relationships between HSVd and its different hosts, HSVd variants from eight naturally infected Prunus sources, including apricot, peach and Japanese plum have been cloned and sequenced. In total, ten molecular variants of HSVd have been identified, nine of which have not been described before. A detailed phylogenetic analysis of the existing HSVd sequences, including the new ones from Prunus determined in this work, points towards a redefinition of the grouping of variants of this viroid, since two new groups were identified, one of them composed of sequences described here. A bias for the presence of certain sequences and/or structures in certain hosts was observed, although no conclusive host-determinants were found. Surprisingly, our analysis revealed that a number of HSVd isolates probably derived from recombination events and thatthe previous hop-type group itself is likelyto be the result of a recombination between members of the plum-type and citrus-type groups.
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Characterization of genotype-specific epitopes of the HN protein of mumps virus
More LessThe SBL-1 strain of mumps virus, grouping with genotype A on the basis of the small hydrophobic protein gene sequence, was grown in the presence of three different monoclonal antibodies. The monoclonal antibodies were directed against the haemagglutinin-neuraminidase (HN) protein and they inhibited haemagglutinating activity and infectivity of the virus. The HN genes of the nine neutralization-escape virus mutants were sequenced and the predicted amino acid sequences were compared with that of the parental virus. Amino acid substitutions were found at positions 269, 352 and 354, respectively, of the 582 amino acid long protein. The three monoclonal antibodies did not react with 35 virus strains isolated in Stockholm during the years 1970 to 1985. Thirteen and four of the strains were found to belong to the D and C genotypes, respectively. A type-specific neutralization antibody response was also found in sera of rabbits hyperimmunized with purified virions of genotype A and D. The genotype-specific difference in neutralizing activity in mice and rabbits was not corroborated by an overall difference in the amino acid sequence of the HN protein of the different genotypes. Further studies are needed to explore the efficacy of mumps virus vaccines for protection against homologous and heterologous genotypes of mumps virus.
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Recombinant vaccinia viruses expressing the F, G or N, but not the M2, protein of bovine respiratory syncytial virus (BRSV) induce resistance to BRSV challenge in the calf and protect against the development of pneumonic lesions
The immunogenicity and protective efficacy of recombinant vaccinia viruses (rVV) encoding the F, G, N or M2 (22K) proteins of bovine respiratory syncytial virus (BRSV) were evaluated in calves, the natural host for BRSV. Calves were vaccinated either by scarification or intratracheally with rVV and challenged 6 to 7 weeks later with BRSV. Although replication of rVV expressing the F protein in the respiratory tract was limited after intratracheal vaccination, the levels of serum and pulmonary antibody were similar to those induced following scarification. The serum antibody response induced by the F protein was biased in favour of IgG1 antibody, whereas the G and the N proteins induced similar levels of IgG1:IgG2, and antibody was undetectable in calves primed with the M2 protein. The F protein induced neutralizing antibodies, but only low levels of complement-dependent neutralizing antibodies were induced by the G protein, and antibody induced by the N protein was not neutralizing. The F and N proteins primed calves for BRSV-specific lymphocyte proliferative responses, whereas proliferative responses were detected in calves primed with the G protein only after BRSV challenge. The M2 protein primed lymphocytes in only one out of five calves. Although there were differences in the immune responses induced by the rVVs, the F, G and N, but not the M2, proteins induced significant protection against BRSV infection and, in contrast with the enhanced lung pathology seen in mice vaccinated with rVV expressing individual proteins of human (H)RSV, there was a reduction in lung pathology in calves.
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Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells
More LessA field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita- MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK2) cells. MVC11 was adapted to LLC-MK2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8 × 105 cell-infecting units (CIU) per mouse. On the other hand, with an inoculum of only 4 × 101 CIU per mouse, corresponding to 1 LD50, M1 replicated well in mouse lung and was highly virulent to mice. Nucleotide and deduced amino acid sequence analyses of the entire genomes of M1 and MVC11 revealed that adaptation to LLC-MK2 cells and the attenuation of mouse pathogenicity of MVC11 were associated with only two amino acid substitutions; one on the C protein (Phe substituted by Ser at position 170) and the other on the RNA polymerase, the L protein (Glu substituted by Ala at position 2050).
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Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro
Acute measles is associated with pronounced immunosuppression characterized both by leukopenia and impaired lymphocyte functions. In an earlier study, we found that mitogen-dependent proliferation of uninfected human peripheral blood lymphocytes (PBLs) and spontaneous proliferation of human cell lines of lymphocytic or monocytic origin was impaired after contact with UV- inactivated, measles virus (MV)-infected cells, UV- inactivated MV or with cells transfected with MV glycoproteins (gp) F and H. We now show that mitogen-stimulated PBLs and Jurkat cell clones either highly sensitive or resistant to CD95-induced apoptosis have a similar sensitivity to MV-induced inhibition and do not undergo apoptosis. Moreover, unimpaired mitogen-dependent upregulation of important activation markers, including IL-2R, was observed in PBL cultures after contact with MV- infected, UV-irradiated presenter cells. This indicates that the cells were indeed viable and acquire a state of activation. Less IL-2 was released from PBLs after contact with MV-infected presenter cells when compared with that released after contact with uninfected cells. However, mitogen-induced proliferation of PBLs was not restored by addition of IL- 2 under these conditions. It appeared that a higher fraction of mitogen-stimulated PBLs accumulated in the G0/G1 phase of the cell cycle after contact with MV-infected cells. Thus, the mitogen- unresponsiveness of PBLs seen after contact with MV-infected cells is due to cell cycle arrest rather than apoptosis.
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Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model
More LessA synthetic peptide representing residues 397–420 from the measles virus (MV) fusion (F) protein was tested for its structure, immunogenicity and protective capacity against intracerebral challenge with a neuroadapted strain of MV. Analysis of the peptide by mass spectrometry showed that it was linear, despite the presence of two cysteine residues in the sequence. Circular dichroism spectroscopy highlighted a weak preference for the peptide to adopt an α-helical conformation. The peptide was shown to be immunogenic in BALB/c and C57BL/6 mice after intraperitoneal immunization in Freund’s adjuvant, and anti-peptide antibodies from both strains of mice reacted with the MV as a solid phase antigen on an ELISA plate. When the fine specificity of the anti-peptide antibody response was examined using overlapping 8-mer peptides, serum antibodies from BALB/c mice recognized the region between residues 407 –417 whereas antibodies from C57BL/6 mice recognized the region 408 –420 of the 397–420 peptide sequence. Although anti-397–420 antibodies had no demonstrable neutralizing activity, protection against challenge with a neuroadapted strain of MV was demonstrated following active immunization with peptide in C57BL/6 mice or after passive transfer of anti-peptide antibodies in BALB/c mice. These findings highlight the importance of the 397–420 region in the induction of protective antibodies in the MV encephalitis mouse model, and suggest that this epitope might be a good candidate for inclusion in a future MV synthetic peptide vaccine.
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Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides
More LessIntracellular transport, glycosylation, tetrameriza- tion and enzymatic activity of the neuraminidase (NA) of fowl plague virus (FPV) were analysed in vertebrate cells after expression from a vaccinia virus vector. Tetramerization occurred with a halftime of 15 min, whereas passage through the medial Golgi apparatus and transport to the plasma membrane occurred with half-times of 2 and 3 h, respectively, suggesting a step in NA maturation beyond tetramerization that limits the rate of transport to the medial Golgi. NA transport rates were about fourfold slower than those of haemagglutinin (HA). Slow transport and processing of FPV NA was not altered by coexpression of FPV HA, nor was the transport rate of HA influenced by NA. The slow transport kinetics of NA were also observed in FPV- infected CV-1 cells. As deduced from the coding sequence, FPV NA has the shortest stalk of all naturally occurring NAs described to date and contains only three potential N-glycosylation sites, which are all located in the globular head domain. Elimination of each of the three N-glycosylation sites revealed that the two oligosaccharides at positions 124 and 66 are of the complex type, whereas the one at Asn-213 remains in mannose- rich form. The glycosylation mutants showed also that oligosaccharides at positions 124 and 213 of FPV NA modulate enzymatic activity. Transport of NA is not influenced by single elimination of any of the three oligosaccharide attachment sites. Mutational analysis of the three Cys residues not involved in intrachain disulfide pairing revealed that Cys-49 in the stalk of the NA molecule is responsible for the formation of disulfide-linked dimers. Analysis of cysteine mutants of FPV NA also demonstrated that disulfide-linked dimers are not absolutely necessary for the formation of enzymatically active tetramers but may stabilize the quaternary structure of NA.
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Identification of a single genotype of hepatitis G virus by comparison of one complete genome from a healthy carrier with eight from patients with hepatitis
More LessDifferent isolates of a putative hepatitis virus called hepatitis GB virus C or hepatitis G virus (HGV) have been cloned recently from patients with hepatitis. This virus has also been found commonly in healthy carriers. We have cloned and sequenced a complete HGV genome, designated HGVCN, from a healthy Chinese blood donor. HGVCN shares 85·8–90·0% nucleotide sequence identity and 95·4–97·5% amino acid identity with the eight available full- length HGV genomes. Furthermore, the majority (82·8%) of the nucleotide substitutions found in HGVCN were synonymous and a fairly uniform distribution of changes was found across the entire genome without identification of any hypervariable region. When compared with the African isolates GBVC and GBVC-EA, the HGVCN-encoded polyprotein contained a 31 amino acid N-terminal extension which was predicted to be a defective core-like sequence. The sequences of the HGV E1 and E2 proteins displayed unique motifs and were highly conserved. Phylogenetic analysis revealed that all nine complete HGV isolates were closely related and that HGVCN grouped with the other Chinese HGV isolate (HGVC964). Taken together, our findings suggest that there is one single genotype of HGV and that the HGV genome cloned from the healthy carrier is not significantly different from those derived from patient sera.
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Molecular analysis of the differential restriction of human immunodeficiency virus type 1 replication in neuronal cell lines
More LessHuman immunodeficiency virus type 1 (HIV-1) replication is restricted partially in SK-N-MC and completely in SK-N-SH neuronal cells. To investigate the molecular mechanism of this differential restriction of HIV-1 replication, cells infected with HIV-1 were analysed for their steady-state levels of: total and unintegrated HIV-1 DNA by DNA PCR, different species of HIV-1 RNA by RT-PCR, and HIV-1 p24 protein production by an ELISA procedure. We found that the kinetics of the infection were slower and there was a lower level of accumulation of HIV- 1 macromolecules (total and unintegrated circular DNA, unspliced and spliced RNAs and viral proteins) in the SK-N-MC cells than in the permissive CEM cells. In SK-N-SH cells, HIV-1 DNA was only transiently detected during the first 24 h post-infection, and the unspliced RNA was detected up to 1 week post-infection. However, the HIV-1 spliced RNAs and the 2-LTR circular DNA were not detected at all during the course of infection. Both SK-N-MC and SK-N-SH cells showed higher levels of HIV-1 DNA, RNA and p24 protein when infected with an HIV-1 (amphotropic retrovirus) pseudotype, HIV-1B. However, the level of HIV-1 replication was still lower in SK-N-SH than in SK-N-MC cells. Moreover, although the kinetics of viral protein production were comparable in SK-N-MC cells infected with HIV-1B and CEM cells infected with HIV-1, the overall level of virus replication was still much lower in HIV-1B- infected SK-N-MC cells. These data suggest that the restriction of HIV-1 replication in neuronal cell lines takes place at both virus-entry and post-entry levels, and cellular factors may be involved in the differential restriction of HIV-1 replication in these cells.
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Salmonella typhimurium aroA recombinants and immune-stimulating complexes as vaccine candidates for feline immunodeficiency virus
Two experimental feline immunodeficiency virus (FIV) vaccines were tested, either alone or in combination, in four groups of cats (A-D). One vaccine (SL3261-FIV) was composed of live attenuated Salmonella typhimurium aroA (SL3261) strains expressing the capsid (Gag) and part of the envelope (Env) proteins of FIV. The other was composed of FIV Gag and Env proteins incorporated into immune- stimulating complexes (iscom-FIV). Cats of group A were immunized four times with SL3261-FIV. Cats of group B were immunized twice with SL3261-FIV and then twice with iscom-FIV. Cats of group C were immunized twice with SL3261 expressing the B subunit of cholera toxin (SL3261-CtxB) and then twice with iscom-FIV. Cats of group D, which served as negative controls, were immunized twice with SL3261-CtxB and then twice with iscom into which the Gag and Env proteins of simian immunodeficiency virus (SIV) had been incorporated (iscom- SIV). Two weeks after the last immunization, all cats were challenged with FIV. At this time, cats immunized with iscom-FIV (groups B and C) showed strong plasma antibody responses to Gag and Env, whilst these responses were weak or undetectable in the cats immunized four times with SL3261-FIV (group A). Seven weeks after FIV challenge, Env-specific antibody responses had increased considerably in cats of all groups except group A. The mean virus loads in the cats of this group proved to be lower than those of the other groups at all time points, indicating partial protection.
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Role of the Fas/Fas ligand pathway in apoptotic cell death induced by the human T cell lymphotropic virus type I Tax transactivator
More LessTwo distinct human diseases have been described in association with human T cell lymphotropic virus type I (HTLV-I) infection: adult T cell leukaemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Although comprehensive understanding of specific mechanisms underlying pathogenesis of either disease has not yet been achieved, the viral regulatory protein Tax is believed to play a significant role. Previous studies demonstrated the potential of Tax to transform host cells. Here, it is shown that the Tax transactivator has in addition the potential to induce T cell death by apoptosis. Using an inducible system (Jurkat cell line JPX-9), significant apoptotic cell death upon Tax expression was observed. In an attempt to detect the cellular genes mediating this effect, it was found that induction of Tax was associated with marked up- regulation of the Fas ligand (FasL) gene. Tax- induced apoptosis was inhibited when the Fas/FasL pathway was interrupted by YVAD-cmk,the inhibitor of ICE-like proteases. Transient expression experiments provided additional support for the putative role of endogenous FasL in Tax-induced apoptosis. Upon cotransfection with Tax-expressing plasmid, the transcriptional activity of the FasL promoter was found to be significantly upregulated in Jurkat cells and several other cell lines, as measured by reporter gene expression. Furthermore, cotransfection using different Tax mutants demonstrated that both CREB and NF-k -B activation domains of Tax protein were required for the transactivation to take effect.
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Regulatory interactions of transcription factor YY1 with control sequences of the E6 promoter of human papillomavirus type 8
More LessHuman papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Our study shows that sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nt 175–179) and comprising the presumable CCAAC and TATA boxes of the E6 promoter P175 contain a cluster of four motifs similar to the DNA recognition site of the multifunctional cellular transcription factor yin-yang 1 (YY1). Using DNase I footprinting and gel retardation tests it could be demonstrated that three of these motifs indeed act as YY1 binding sites. To test their functional relevance for P175 activity, engineered YY1 binding site mutants were analysed in the context of a P175 test vector using transient expression assays with human keratinocytes. YY1 turned out to exert both positive and negative effects upon the activity of the HPV-8 E6 promoter; binding of YY1 to a site upstream of the promoter’s cap-position (BS1) activated transcription, whereas the downstream site (BS2) mediated repression. The second downstream YY1 binding site (BS3) seemed to play an auxiliary role, enhancing the negative effect of YY1 BS2. These observations define YY1 as an important cellular protein involved in the control of E6 oncogene expression of the skin-specific ‘high risk’ HPV-8 and emphasize the potential regulatory role of sequences located downstream of the transcription start site.
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Preferential virosomal location of underphosphorylated H5R protein synthesized in vaccinia virus-infected cells
More LessThe phosphorylation state of vaccinia virus (VV) protein H5R synthesized in infected cells was investigated by two-dimensional gel electrophoresis. Most of the H5R protein was underphosphorylated (pI 5 9 to 6 8) and, on centrifugation of cell lysates, was associated with virosomes sedimenting with nuclei. However, about a quarter of the H5R protein synthesized was highly phosphorylated (pI 5 5), and this was the major form of the H5R protein present in cytoplasmic extracts. Immunofluorescence of VV- infected cells in the absence of DNA replication showed that underphosphorylated H5R protein, specifically recognized by antibody, was abundantly distributed throughout the cytoplasm but also present in punctate particles, whereas most of the B1R protein detected was in the punctate particles. Late gene expression was not required for the H5R protein to accumulate in virosomes-viral DNA synthesis was sufficient. The different phosphorylation states and cytological locations of the H5R protein suggest it has multiple roles in VV development.
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Effective priming of neonates born to immune dams against the immunogenic pseudorabies virus glycoprotein gD by replication-incompetent adenovirus-mediated gene transfer at birth
More LessOne of the main limitations of the vaccination of neonates from vaccinated or infected mothers is the interference by inherited maternal antibodies, which are known to inhibit the immune response against both live and inactivated vaccines. The efficiency of bypassing this inhibition by the transfer of an immunogenic glycoprotein gene, the gD gene of pseudorabies virus (PRV), into neonates was explored. The experiments were conducted in 1- day-old piglets, which are immunocompetent at birth. The same transcription unit (gD of PRV under the control of the adenovirus major late promoter) was delivered intramuscularly at birth either in the form of naked DNA or cloned in the genome of a replication-defective adenovirus. A booster injection of a conventional live PRV vaccine strain was given at 10 weeks of age, the replication of which was greatly restricted by the residual amounts of colostral antibodies in control animals. Piglets were challenged at the age of 16 weeks with a virulent PRV strain. The replication-defective adenovirus was able to efficiently prime piglets born to immune dams against gD in such a way that inoculation with the Bartha strain protected them against a subsequent challenge with the same level of efficacy in piglets born to naive or immune dams. In contrast, piglets born to immune dams into which the gD gene was not transferred, or transferred as naked DNA at birth, were not protected. These results open the way for early immunization of neonates born to vaccinated or infected mothers.
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Biologically safe, non-transmissible pseudorabies virus vector vaccine protects pigs against both Aujeszky′s disease and classical swine fever
More LessEnvelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not required for cell-to-cell spread. When animals are inoculated with a phenotypically complemented PRV gD mutant, the virus is able to spread locally by means of direct cell-to-cell transmission, but progeny virions released by infected cells are non-infectious because they lack gD. Therefore, the virus cannot be transmitted from inoculated animals to other animals. This property makes a PRV gD mutant an attractive candidate as a safe vaccine vector. To examine whether a self-restricted, non-transmissible PRV mutant can be used as a biologically safe vaccine vector, a gD/gE-negative PRV recombinant virus which expresses envelope glycoprotein E2 of classical swine fever virus was constructed. Vaccination of pigs showed that the recombinant virus was able to protect pigs against both Aujeszky’s disease and classical swine fever.
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Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1
More LessImmunocytochemistry on serial paraffin sections was used to monitor the production dynamics of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α) and viral antigens in the trigeminal ganglion (TG) and the central side of the dorsal root entry zone (DRE) of mice, following infection of the cornea with herpes simplex virus type 1. In normal TG, scattered satellite cells were TNF-α and in the DRE, TNF-α and/or low numbers of IL-6 cells were detected. On day 3 after infection, foci of TG neurons with viral antigens were surrounded by large numbers of TNF-α and/or IL-6 cells and low numbers of IFN-γ cells. IL-2 and/or IL-4 cells appeared later, when viral antigens had almost cleared. In the TG, the most striking changes occurred with TNF-α, with respect to its source (satellite cells, Schwann cells and infiltrating cells) and the extent and long duration of its production. TNF-α was the predominant cytokine throughout acute and latent infection and even by day 30, numbers of satellite cells expressing this cytokine were three times higher than those in normal ganglia. Moreover, in the DRE, TNF-α was the only cytokine detected during virus clearance and again, its production continued, along with that of IL-6, on days 20 to 30, in both infiltrating cells and astrocytes. Thus, cytokines, particularly TNF-α and perhaps IL-6,from infiltrating cells and resident glial cells may have a role both in virus clearance and in normal homeostatic mechanisms in the nervous system such as repair and protection of neurons from damage.
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Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm
More LessHerpes simplex virus type 1 (HSV-1) immediate early protein IE63, an essential nuclear protein, is pleiotropic in function and, at the post-transcriptional level, inhibits RNA splicing, interacts with cellular splicing small nuclear ribonucleoprotein particles (snRNPs), binds RNA and prevents the nucleocytoplasmic transport of intron-containing mRNAs. Here it is reported that IE63 is a nucleo-cytoplasmic shuttle protein able to travel from snRNP-and RNA-rich nuclear foci to the cytoplasm, where it accumulates during actinomycin D treatment. This newly identified property suggests that IE63 facilitates nuclear export of HSV-1 transcripts, in addition to retaining intron-containing transcripts in the nucleus. The mechanism by which IE63 controls RNA export has yet to be defined.
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Herpes simplex virus 1716, an ICP 34.5 null mutant, is unable to replicate in CV-1 cells due to a translational block that can be overcome by coinfection with SV40
More LessHerpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell- induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV- 1716 is unable to replicate in the simian kidney cell- derived line CV-1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV- 1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild-type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non-permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2α) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.
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Analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection
More LessSmall DNA viruses (adenoviruses, simian virus 40, or human papillomaviruses) induce S-phase progression but prevent cell division to provide precursors for viral DNA replication. Herpes simplex viruses types 1 or 2 (HSV-1 or HSV-2) contain genes which encode DNA-metabolizing enzymes, for example, ribonucleotide reductase, thymidine kinase and dUTPase, suggesting that S-phase factors are not required for an efficient infection. However, several studies indicated that HSV induces some events that occur during cell-cycle progression. To determine if HSV-2 induces S-phase entry, we examined serum-arrested African green monkey kidney cells (CV-1) after infection. Two hours after infection steady-state levels of the S-phase-specific cyclin, cyclin A, increased. S-phase cyclin-dependent kinase activity (CDK2) was stimulated 10-fold 8 h after infection but decreased at 16 or 24 h after infection. Mitotic CDK activity (CDC2) was not activated after infection, in part due to decreases in CDC2 protein levels and inactivation of enzymatic activity resulting from tyrosine phosphorylation of CDC2. Furthermore, CDK4 activity was not dramatically affected by infection. These studies indicate that HSV-2 infection selectively activates CDK2 after infection but cell-cycle progression does not occur. We hypothesize that infection activates certain components of the cell cycle which enhance viral gene expression and DNA replication.
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Development of a model for cytomegalovirus infection of oligodendrocytes
More LessThe well-characterized human oligodendroglioma (HOG) cell line, cells of which resemble immature oligodendrocytes, was used to investigate the level of permissiveness to human cytomegalovirus (CMV) infection. Expression of CMV genes was incomplete following exposure of HOG cells to CMV, in contrast with results observed with the astroglioma cell line U373-MG (used as a positive control). However, treatment with phorbol 12-myristate 13-acetate (PMA) or with dibutryl cAMP (dbcAMP) plus the phosphatase inhibitor 1-isobutyl-3,3-methyl xanthine (IBMX) rendered the HOG cells fully permissive to CMV; down-regulation of HLA class I and production of virions were only observed under these conditions. In contrast to the findings seen with the HOG cell line, treatment of U373-MG cells with dbcAMP/IBMX or PMA did not interfere with CMV- induced down-regulation of HLA class I. However, these chemical stimulators reduced virus production in U373-MG cells by 30 and 70%, respectively.
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PCR amplification is more sensitive than tissue culture methods for Epstein-Barr virus detection in clinical material
More LessIn this study we have compared the use of PCR and conventional tissue culture methods to detect Epstein-Barr virus (EBV) in peripheral blood mononuclear cells and throat wash samples. The study population included 29 healthy adult and 20 immunocompromised EBV-seropositive donors. The results show significantly higher EBV detection rates by PCR than the tissue culture methods in throat wash samples from both donor groups (P < 0·01 in healthy donors and P < 0·009 in the immunocompromised donors) and in peripheral blood from the immunocompromised but not from the healthy donors (P < 0·008). Furthermore, when EBV DNA detection rates in throat wash cell pellet and supernatant fluid were compared, a higher positive result was obtained with the cell pellets which reached statistical significanceinthe immunocompromised group (P < 0·02). No correlation was found between positivity in throat wash and peripheral blood from the same donors.
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