- Volume 78, Issue 1, 1997
Volume 78, Issue 1, 1997
- Articles
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The cleavage activities of aphthovirus and cardiovirus 2A proteins.
More LessThe primary 2A/2B polyprotein cleavage of aphtho- and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/β-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (~85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler’s murine encephalomyelitis virus (TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency - if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-ΔTME2A-GUS] polyprotein was able to mediate cleavage with high (~85%) efficiency-directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-Δ EMC2A- GUS] polyprotein which also mediated cleavage at ~85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.
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Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus
More LessNatural resistance to flaviviruses in mice is controlled by a single genetic locus, Flv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flvs ) and -resistant congenic C3H/RV (Flvr ) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0·6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flvr interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.
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Characterization of monoclonal antibody-escape mutants of tick-borne encephalitis virus with reduced neuroinvasiveness in mice
More LessEscape mutants of tick-borne encephalitis (TBE) virus were selected using neutralizing monoclonal antibodies (MAbs) that react with three different and previously unrecognized epitopes in the envelope protein E of TBE virus. Two of these variants (V-IC3 and V-IE3) exhibited a significantly reduced reactivity with their selecting MAbs, as determined by ELISA, whereas with one variant (V-IO3), reactivity was completely unchanged. Comparative sequence analyses demonstrated that each of the variants differed from the wild-type virus by a single amino acid substitution located at exposed positions within domains I, II and III of protein E. In the mouse model, all three mutants were still neurovirulent but exhibited a significantly reduced neuroinvasiveness after subcutaneous inoculation. Virus replication, however, was sufficient to induce a specific antibody response. The observed alterations in virulence properties were not associated with reduced growth rates in vertebrate cell cultures, but one variant (V-IE3) exhibited a small plaque phenotype. The mutation of variant V-IO3 resulted in a temperature-sensitive phenotype and a significant elevation of the pH-threshold of the conformational change necessary for fusion activity.
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Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae
cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1–181 of NS3; NS3181) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3181 protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3181 protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3181 protease will facilitate the development of anti-hepatitis C therapies.
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Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences
More LessThe phylogenetic status of recently described isolates of hepatitis C virus (HCV) from Vietnam, Thailand and Indonesia (previously classified as types 7, 8, 9, 10 and 11) was re-analysed by the neighbour-joining method instead of the unweighted pair-group method with arithmetic mean (UPGMA) that was first used by the discoverers of these strains. The analysis of complete amino acid sequences and of nucleotide sequences of the envelope 1 (672 nt) and nonstructural 5B (1092 nt) genomic regions permitted the re-assignment of the type 7, 8, 9 and 11 isolates to type 6, and that of type 10 strains to type 3. Finally, this study made possible the classification of the previously described HCV strains (including these South-East Asian isolates) in six major types and at least 30 subtypes. It confirms that analysis of the E1 and NS5B genomic regions using the neighbourjoining method is a reliable tool for the assignment of most new isolates.
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Genetic diversity between hepatitis G virus isolates: analysis of nucleotide variation in the NS-3 and putative ‘core’ peptide genes
More LessSignificant variation was found, between 46 isolates of hepatitis G virus (HGV), following direct sequencing of subgenomic PCR fragments from either or both the NS-3 and putative ‘core’ peptide. Nucleotide sequences of most HGV NS-3 fragments varied by 10–30% and of most putative ‘core’ peptide fragments by 2–20%. HGV was therefore shown to be much less variable than hepatitis C virus (HCV) and pairwise comparisons ofHGVsequencesdemon- strated a single distinct distribution of evolutionary distances. Construction of phylogenetic trees, bootstrap analysis and calculation of mean distances between possible subtypes also indicated one level of variation between HGV NS-3 and putative ‘core’ peptide sequences, and the suggested degree of variation between isolates was similar to that between HCV subtypes. No evidence for clustering of sequences into multiple subtypes or genotypes was found. Although very small subgenomic fragments of HCV are indicative of the viral genotype it seems that the assignment of genetic groups is not possible for HGV using such small subgenomic fragments. The relatively limited genetic variation observed in HGV may reflect a relatively low level of host selection pressure stemming from the low level of host immunity stimulated by this virus.
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Mouse hepatitis virus strain A59 is released from opposite sides of different epithelial cell types
More LessCoronaviruses infect humans and animals through epithelial cells of the gastrointestinal and respiratory tracts that serve as their primary target. When studying infections in cultured polarized epithelial cells, we found previously that coronaviruses are released from specific plasma-membrane domains; thus, mouse hepatitis virus (strain A59; MHV-A59) leaves murine epithelial kidney cells from the baso- lateral surface, whereas release of transmissible gastroenteritis virus from porcine epithelial kidney cells is confined to the apical membrane. This observation begged the question whether a particular coronavirus is consistently shed through the same membrane, irrespective of the nature of the epithelial cell. We therefore extended our studies with MHV-A59 to Madin-Darby canine kidney (MDCK) strain I and human colon carcinoma (Caco-2) cells, both of which are naturally refractory to MHV-A59 but were made susceptible to infection by transfection with recombinant MHV receptor cDNA. The release of MHV-A59 from CacoMHVR cells occurred preferentially from the basolateral side, consistent with our previous observations. In contrast, release from MDCK mhvr cells occurred almost exclusively from the apical surface. Because of this difference, we studied MHV-A59 infection of MDCKmhvr cells in more detail. The virus entered the cells preferentially from the apical side, a situation similar to that in murine epithelial cells, where the highest density of MHV receptor glycoprotein was found. The results from this and previous studies show that targeting of vesicles containing MHV-A59 to a specific side of epithelial cells may vary in different epithelial cell types.
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Expression and characterization of a recombinant murine coronavirus 3C-like proteinase
More LessThe replication of coronaviruses involves proteolytic processing of the gene 1 translation products, pp1a and pp1ab. One of the key enzymes in this process is predicted to be a virus-encoded 3C-like proteinase. In this report, we describe a bacterial system that has allowed us to express and characterize a recombinant murine coronavirus (MHV- JHM) 3C-like proteinase. The partially purified protein has been shown to exhibit proteolytic activity in trans and mutation analysis has been used to demonstrate the indispensability of Cys- 3495 for enzymatic activity. Finally, the effect of class-specific proteinase inhibitors on the trans cleavage activity of the MHV 3C-like proteinase has been used to demonstrate the functional and structural homology of this enzyme to the picorna- virus 3C proteinases.
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Organization of the M genomic segment of Toscana phlebovirus
More LessThe nucleotide sequence of the Toscana (TOS) virus M RNA segment contains a single major open reading frame in the viral-complementary sequence, which can encode a polyprotein of 1339 amino acids. To map the TOS M segment product(s), different regions of the putative M polypeptide were expressed as glutathione S-transferase fusion proteins, which were purified and inoculated into mice to produce hyperimmune sera. By Western blot analysis, a protein of approximately 30 kDa and two glycoproteins, G1 and G2, with the same molecular mass (approximately 65 kDa) were identified in TOS virus-infected cells. The 30 kDa protein, which reacted with antibodies raised to the NH2-terminal, was found to be a non-structural protein (designated NSm). By immunoprecipitation analysis of TOS virus-infected cell lysates, both treated or untreated with tunicamycin, the relative positions of glycoproteins G1 and G2 were determined. The gene order, with respect to the genomic M RNA, was found to be 3′ NSm-G1-G2 5′.
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An anti-fusion regulatory protein-1 monoclonal antibody suppresses human parainfluenza virus type 2-induced cell fusion
Fusion regulatory protein-1 (FRP-1) regulates virus-mediated cell fusion and induces polykaryocyte formation of monocytes without any fusogen. We have recently reported that FRP-1 and the 4F2/CD98 heavy chain are identical molecules. Cell fusion in Newcastle disease virus (NDV)-infected HeLa cells was enhanced when cells were incubated with anti-FRP-1 MAb. Anti-FRP-1 MAbs also induced human immunodeficiency virus gp160-mediated cell fusion. However, HBJ127, an anti-FRP- 1/4F2/CD98 MAb that enhanced cell fusion in NDV- infected cells, delayed human parainfluenza virus type 2 (HPIV-2)-induced cell fusion in HeLa cells, although these viruses belong to the same genus Rubulavirus. No anti-FRP-1 MAbs enhanced cellfusion in HPIV-2-infected HeLa cells. Anti-FRP-1 MAbs including HBJ127 showed no effect on virus growth and expression levels of virus-specific polypeptides in HPIV-2-infected HeLa cells, indicating thatthe delay in cell fusion by an anti-FRP-1 MAb is not due to suppression of virus replication. When HeLa cells were transfected with an expression vector harbouring HPIV-2 HN and F genes, cell fusion was also suppressed by HBJ127, but the effect was weak in comparison with virus-infected cells. These data indicate anti-FRP-1 antibodies not only induce/enhance, but also inhibit/delay virus- induced cell fusion and therefore FRP-1 molecules are multifunctional.
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Characterization of five conserved genotypes of the mumps virus small hydrophobic (SH) protein gene.
More LessTwenty-one different mumps virus isolates from Sweden and Japan collected over 25 years were compared by nucleotide sequence analysis of the small hydrophobic (SH) protein gene, and the deduced 57 amino acid sequences of the coding part of the gene were aligned with published sequences of viral isolates from the USA, the UK, Sweden and Japan. Five genotypes were found which, in accordance with previously used nomenclature, were named A to E. Genotypes A, C, D and E were found in Europe and genotype B was found in Japan. Amino acid signature sequence motifs specific for each genotype were identified. A triplet of three amino acids at positions 28-30 was the most characteristic. Different genotypes can circulate simultaneously in a given geographical location. In Stockholm, genotypes A and D or C and D were found over different time periods. In contrast, only genotype B was found in Japan.
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Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture.
Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 × 10−4 per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.
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Inhibition of measles virus infection and fusion with peptides corresponding to the leucine zipper region of the fusion protein.
More LessMeasles virus (MV) infections are characterized by the induction of syncytia, i.e. the fusion of infected cells. Two MV proteins, the haemagglutinin (HA) and fusion (F) proteins, are involved in this process. Synthetic peptides representing two α-helical regions of the MV F protein were studied for their ability to inhibit MV fusion. A peptide corresponding to the leucine zipper region (amino acids 455–490) inhibited MV fusion, whereas a peptide to amino acids 148–177, corresponding to the amphipathic α-helix region, did not. Fusion inhibition was also obtained with vaccinia virus-expressed HA and F, a recent wild-type MV isolate and the closely related canine distemper virus, but not with mumps virus. The F455–490 peptide did not affect the synthesis of MV F or its transport to the cell membrane. Virus-cell attachment was unaffected, but haemolysis and virus entry into the cell were inhibited. In one-step growth curves the virus yield was unaffected.
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Distribution and variation of NV genes in fish rhabdoviruses
More LessThe fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) contains a non-virion (NV) gene between the glycoprotein (G) and polymerase (L) genes on its RNA genome. The present study investigated three other fish rhabdovirus genomes and found that the NV gene of hirame rhabdovirus is closely related to the NV of IHNV, whereas the viral haemorrhagic septicemia NV gene showed evidence of significant divergence. Most importantly, spring viraemia of carp virus, the only vesiculovirus-like fish rhabdovirus examined, did not have an NV gene at its genomic RNA G-L junction. These results suggest that the presence of an NV gene is characteristic of the unassigned fish rhabdovirus subgroup previously classified as lyssaviruses, and that the NV gene is not essential for replication in fish cells per se, since it is absent in a vesiculovirus-like fish rhabdovirus.
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Mapping of monoclonal antibody epitopes of the rabies virus P protein
More LessThirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E. coli. All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells. The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells. Western blotting, immunoprecipitation and immunofluorescence assays were performed. These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region. Most of the antigenic sites appeared to be composed of linear epitopes. These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.
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Inhibition of vesicular stomatitis virus in cells constitutively expressing an antisense RNA targeted against the virus RNA polymerase gene
More LessTo study the effect of virus-specific antisense RNA expression on vesicular stomatitis virus (VSV) in-fectivity in cultured cells, a HeLaS3 cell line constitutively expressing antisense RNA complimentary to a portion of the VSV large RNA-dependent RNA polymerase gene (L) was established (HeAntiL). At an m.o.i. of 0·01 or 0·1, the HeAntiL cell line was able to reduce virus titre and delay virus-induced cell death by 9 or 5 h, respectively, when compared to a HeLa cell line stably transfected with the expression vector devoid of antisense sequence. Ribonuclease protection experiments showed a 10–20-fold reduction of hybridizable virus L mRNA in infected HeAntiL cells compared to infected control cells at various times before cell death. These results indicate that the antisense RNA approach can significantly reduce VSV mRNAtran-scription and virus production for a reasonable period of time. The robust growth rate of VSV eventually overwhelms the available antisense RNA and leads to delayed cell death.
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Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells.
More LessThe activity in vivo of HIV-1 proteinase (PR) was analysed in the baculovirus expression system, using eight different constructs of the prt gene under the control of polyhedrin (PH) promoters of various strengths. None of the active PRs was expressed in substantial quantities, and only PH-fused and/or non-functional PR mutants accumulated in high amounts in insect cells. However, enough PR activity was generated from a lengthened PR construct in insect cells to process Gag polyprotein substrate co-expressed in the same cells in trans. Fusion of the first 58 residues from the PH sequence to the PR N terminus did not significantly change its activity and specificity of cleavage of the Gag substrate. When analysed under mild denaturing conditions, PH-fused or unfused full-length PR point mutants, as well as PH-fused or unfused C-terminal deletion mutants, showed a propensity to multimerize, with a predominant occurrence of dimers. The incorporation of PR into Gag particles was studied using eight Gag-PR fusion constructs, all containing a non-functional PR mutant. The PR domain was fused to the C-terminal p6 domain of Gag (p6 gag ), or translated in frame with NCp7 (as in frameshifted Gag-Pol polyprotein) and followed by downstream sequences of increasing lengths from the Pol domain or the bacterial β-galactosidase. The results suggested that the presence of the p6 gag domain was detrimental to the encapsidation of polyprotein-embedded PR.
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Infection of choroid plexus cells by human T cell leukaemia virus type I
More LessVery little is known about the factors that determine the outcome of infection by human T cell leukaemia virus type I (HTLV-I) and the neurotropism of this virus is still a controversial point. In transgenic mice, the HTLV-I LTR is active mainly in the central nervous system (CNS), in parenchyma as well as in ependymal and choroid plexus cells. The latter are of particular interest and could represent the way of entry of the virus into the CNS. In this study we show that primary cultures of sheep choroid plexus can be infected with HTLV-I, leading to characteristic multinucleated syncytial cells containing virus RNA and proteins. HTLV-I p24 Gag protein was detected in the culture medium and the presence of virus particles was observed by electron microscopy 40 days after infection. At this time post-infection HTLV-I could be transmitted to human cord blood cells.
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Long-term persistence of protective immunity in cynomolgus monkeys immunized with a recombinant vaccinia virus expressing the human T cell leukaemia virus type I envelope gene.
To develop effective vaccines against infection with human T cell leukaemia virus type I (HTLV-I), we constructed a recombinant vaccinia virus (WR- SFB5env) synthesizing the HTLV-I envelope (Env) gp46 protein under the control of a strong promoter, termed the ATI hybrid promoter. WR- SFB5env expressed a large quantity of gp46. In cynomolgus monkeys (Macaca fascicularis) immunized with WR-SFB5env, anti-HTLV-I Env antibody, including neutralizing antibody, was induced and remained at a high level until 136 weeks (2 6 years) post-infection (p.i.). These immunized monkeys had HTLV-I Env-specific cytotoxic T lymphocyte activity. At 136 weeks p.i., the immunized monkeys were challenged with an HTLV-I-produc- ing cynomolgus T lymphocyte cell line. Neither HTLV-I antigen nor HTLV-I proviruses were detected in peripheral blood mononuclear cells, lymph nodes or spleens of the WR-SFB5env- immunized monkeys, in contrast to non-immunized control monkeys. These results indicate that a single immunization with WR-SFB5env induced prolonged humoral and cellular immune responses to HTLV-I and protected the monkeys against virus challenge.
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Bovine leukaemia virus-induced lymphocytosis in sheep is associated with reduction of spontaneous B cell apoptosis.
Experimental inoculation of sheep with bovine leukaemia virus (BLV), a retrovirus homologous to the human T-lymphotropic virus type 1 (HTLV-1), induces a chronic expansion of the B lymphocyte population (persistent lymphocytosis) and the development of a B cell leukaemia/lymphosarcoma syndrome. To gain insight into the mechanisms of BLV-induced lymphocytosis, we tested B cell survival capacity and cycling activity in peripheral blood mononuclear cells (PBMCs) from lymphocytotic, asymptomatic and control sheep. Interestingly, B cells from lymphocytotic sheep presented a lower level of spontaneous apoptosis (29%) in ex vivo cultures compared to that obtained with infected asymptomatic (42%) and control (57%) sheep PBMCs. Virus capsid (CA) synthesis was mainly found among surviving B cells and the percentage of CA- producing B cells correlated with the extent of B cell survival, indicating that BLV replication in B lymphocytes may promote protection from cell death. B cell survival was not linked with increases in expression of Bcl-2 mRNA or membrane leukosialin (CD43), although both are documented to be involved in some aspects of the B cell life-span. Finally, cell cycle analyses in freshly isolated PBMCs from lymphocytotic sheep revealed a slightly increased proportion of B cells in S phase compared to controls. Altogether, these data suggest that both BLV-induced B cell proliferation and extended survival are involved in the lymphocytotic stage encountered in BLV infection in sheep.
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Activity of JC virus archetype and PML-type regulatory regions in glial cells.
More LessSequence variations are seen in the JC virus promoter/enhancer in virus taken from progressive multifocal leukoencephalopathy (PML) brains and it has been hypothesized that the variations arise in the host at some point in the development of PML. These rearrangements may be adaptations for enhanced growth in glial cells; if so, transcription or replication levels should differ between archetypal and rearranged PML-type promoters. The archetype and four PML-type promoters were analysed in human glial cells for early and late transcriptional activity in the absence or presence of virus T antigen, and for DNA replication. CAT reporter expression differed within a fivefold range and the archetype was intermediate in strength to the PML-type regulatory regions. The archetype differed from rearranged promoters in that the late promoter was less responsive to T antigen and the shift from early to late activity with T antigen was less pronounced. All five regulatory regions demonstrated similar levels of DNA replicating activity. Rearrangement of the archetype was not required for activity in glial cells, but the potential for differences in the regulation of the late capsid genes was found.
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Casein kinase II phosphorylates bovine papillomavirus type 1 E1 in vitro at a conserved motif.
More LessThe E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATP- dependent helicase activity. The E1 protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped. Examination of the predicted amino acid sequence of all available E1 proteins revealed strong conservation between amino acids 25 and 60 of a motif consisting of a serine residue followed by a stretch of acidic residues. This conserved motif resembled a phosphorylation consensus site for the ubiquitous cellular kinase casein kinase II (CKII). Biochemical and mutational analysis demonstrated that the BPV-1 E1 protein is an in vitro substrate for CKII at the serine within this conserved motif.
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Replacement of the herpes simplex virus type 1 Vmw175 DNA binding domain with its varicella-zoster virus counterpart results in a protein with novel regulatory properties that can support virus growth.
More LessThe alphaherpesviruses encode major immediate early transactivator proteins that are essential for the expression of later classes of viral genes. We have previously shown that the extensive sequence similarity between the herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) members of the family (proteins Vmw175 and VZV140k) extends to function, since a virus which expresses VZV140kin place of Vmw175 is able to grow, albeit at reduced efficiency. We have also shown that the DNA binding characteristics of the isolate d DNA binding domains of Vmw175 and VZV140k are related but distinct. In order to assess whether the different DNA binding properties of the two proteins are responsible for the differences in their individual transcriptional regulatory functions, we constructed a plasmid and an HSV-1 virus in which the VZV140k DNA binding domain coding sequences replace those of Vmw175. The characteristics of the resultant hybrid protein in transfection assays and during virus infection suggest that the nature of the DNA binding domain plays a significant role in the transactivation and repression properties of the Vmw175 family of proteins.
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The abundance of the herpes simplex virus type 1 UL37 tegument protein in virus particles is closely controlled.
More LessThe tegument region of herpes simplex virus type 1 virus particles contains approximately 15 virus- encoded polypeptide species. One of the less abundant species is a 120 kDa protein specified by gene UL37. The abundance of the UL37 protein in infected cells was increased about 20-fold by replacing the native promoter for gene UL37 with the strong immediate early promoter of human cytomegalovirus. This rise in abundance did not induce any detectable increase in the amount of UL37 protein incorporated into virus particles. These data contrast with those previously obtained for a second tegument protein VP22 whose level of incorporation was elevated by increasing its abundance in infected cells. Thus, for the UL37 protein, a mechanism exists to control its abundance in the tegument. Moreover, data obtained with light particles which lack the viral capsid suggest that this controlled incorporation is not directed by interaction with the capsid structure.
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Herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha.
More LessWe have analysed the ability of herpes simplex virus type 2 (HSV-2) to induce nitric oxide (NO) production in resting BALB/c mouse peritoneal macrophages. In most experiments, macrophages produced very small amounts of NO upon infection with HSV-2. Mock virus preparations did not induce NO production, and virus inactivation experiments showed that infectious virus was required. Since interferon-γ(IFN-γ) is the prototype cytokine that is able to induce significant NO production in macrophages, we found it of interest to examine the influence of HSV-2 infection on the IFN-γ-induced NO production. The virus exerted a synergistic effect on the IFN-γ-induced NO release, which was accompanied by induction of the iNOS-gene as revealed by RT-PCR. This effect was largely dependent on the presence of infectious virus particles, since only a minor effect was seen with mock virus and inactivated virus preparations. From experiments with neutralizing antibodies to tumour necrosis factor-α(TNF-α) and IFN-α/β it was concluded that the synergistic effect is dependent on autocrine secretion of TNF-α, which acts as a second signal and synergizes with IFN-γ in NO production.
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Transforming growth factor beta production during rat cytomegalovirus infection.
We analysed the production of transforming growth factor β (TGF-β) during a cytomegalovirus (CMV) infection in a rat model system. Splenocytes from immunocompetent rats infected with rat CMV (RCMV) released increased amounts of TGF-β 1. TGF- β production was also evident in RCMV-infected radiation-immunosuppressed rats; their sera inhibited the interleukin 2-induced proliferation of T cells, which could be restored by anti-TGF-β anti bodies. In addition, TGF-β production could be visualized immunohistologically in the lungs, spleen, liver and bone marrow of radiation-immunosuppressed infected rats. The virus directly induced this cytokine since TGF-β was produced upon RCMV infection in vitro. The induction of TGF-β production may contribute to immunosuppression during CMV infection.
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Establishment of a cell line expressing human parvovirus B19 non-structural protein from an inducible promoter.
Human parvovirus B19 non-structural (NS) protein is supposed to play a major role in B19 replication and transcription, and therefore in B19 pathogenicity. Constitutive expression of NS protein in stable cell lines has failed so far, presumably because of its cytotoxicity. To avoid this cytotoxic effect, we have cloned the NS gene in an Epstein-Barr virus episomal vector under the control of a steroid inducible promoter (5xGRE) and transfected this construction into HeLa cells. We obtained stable cell lines inducibly expressing high level of NS protein, with 50% of the cells demonstrating specific nucleo-cytoplasmic staining. In Western blot analysis, three B19 NS proteins (72, 68 and 60 kDa) were found but a unique NS transcript was detected by Northern blotting. The NS protein expressed in HeLa cell lines was demonstrated to be functional as it trans-activates the B19 P6 promoter. These cell lines might be major tools for further study and characterization of B19 NS protein.
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Sequence of porcine circovirus DNA: affinities with plant circoviruses
More LessThe complete nucleotide sequence (1759 nt) of the ssDNA genome of porcine circovirus (PCV) was determined from a cloned dsDNA replicative form isolated from PCV-infected cells. Sequence analysis detected no significant nucleic acid or protein similarity with another animal circovirus, chicken anaemia virus (CAV) but, surprisingly, the highest protein similarity was obtained between the product of the largest predicted PCV ORF (ORF1; encoding a potential protein of 35·7 kDa) and a putative protein encoded by the plant circovirus banana bunchy top virus (BBTV). High protein similarity was also detected with the other plant circoviruses subterranean clover stunt virus (SCSV) and coconut foliar decay virus (CFDV). This region of protein identity corresponds with the putative plant circovirus replication-associated protein (Rep). The presence of a nonanucleotide sequence at the apex of a potential-stem loop structure, identical to that found in the plant circoviruses CFDV and SCSV and similar (one mismatch) to that found in the plant circovirus BBTV and in the geminiviruses, suggests that rolling-circle replication may operate during PCV DNA replication. These findings show that PCV is unique in that it bridges the gap between animal and plant circoviruses. The taxonomic relationship of PCV with other members of the Circoviridae is discussed.
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Two mRNAs are transcribed from banana bunchy top virus DNA-1.
More LessWe have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3′ RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3′ of a cauliflower mosaic virus 35S promoter and 5′ of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3′ end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3′ of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3′ of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.
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In vivo expression of an overlapping gene encoded by the cucumoviruses.
More LessWe recently reported the molecular characterization and functional analysis of an overlapping gene 2b encoded by RNA 2 of the Q strain of cucumber mosaic cucumovirus (Q-CMV). We show here that the homologous gene encoded by the V strain of tomato aspermy cucumovirus (V-TAV) and the WAII strain of CMV (WAII-CMV), which is in a different subgroup to Q-CMV, is also expressed in vivo by demonstrating the accumulation of the mRNA (RNA 4A) and its protein in infected plants. Interestingly, RNA 4A of V-TAV is encapsidated in virions as found previously for Q-CMV whereas WAII-CMV contains very little RNA 4A in virions. As the 2b gene is conserved in all 10 cucumoviral species or strains sequenced to date and the 2b gene is expressed for three of these viruses, we conclude that the 2b gene is a common feature of the Cucumovirus genus.
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Short and long distance spread of potato leafroll luteovirus: effects of host genes and transgenes conferring resistance to virus accumulation in potato.
More LessPotato leafroll luteovirus (PLRV) movement through phloem of PLRV-resistant potato clones was examined in experiments in which stem pieces were grafted either between infected rootstocks and virus-free susceptible scions or between infected scions and virus-free susceptible rootstocks. These test plants permitted either upwards or downwards virus movement into the susceptible tissue. Resistant potato clones had either host gene-mediated resistance (H-MR) or transgene-mediated resistance (T-NR, conferred by transformation with the PLRV coat protein gene) to PLRV accumulation. The rate of PLRV movement was similar whether stem tissue was taken from H-MR, T-MR or susceptible potato clones. Virus movement through two graft unions began around 7 days after grafting and was generally complete by about 14 to 16 days. Virus movement occurred soon after acquiring functional phloem continuity across grafts as demonstrated by tracing with 6(5)-carboxyfluorescein, a phloem-mobile dye. Most of the delay in virus detection after grafting probably resulted from the time necessary to develop new phloem strands across graft unions; subsequent movement of PLRV was rapid suggesting a passive process. PLRV infection was largely excluded from external phloem bundles in stem tissue of clones with either H-MR or T-MR. This trait was less pronounced as tissue aged. The mechanism limiting PLRV invasion of external phloem bundles of the T-MR clones appears to be similar to that operating in the H-MR clones. Results are discussed in the context of a proposed model of PLRV movement.
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3′-Terminal sequences of the RNA genomes of narcissus latent and Maclura mosaic viruses suggest that they represent a new genus of the Potyviridae.
More LessThe nucleotide sequences of part of the nuclear inclusion body b (NIb) gene, the complete coat protein gene and the 3′ untranslated regions of narcissus latent virus (NLV) and Maclura mosaic virus (MacMV) were determined. Deduced amino acid sequences for the NIb and coat protein genes revealed that NLV and MacMV are closely related. Gel analysis and Western blotting of the coat proteins of NLV and MacMV from infected tissue or purified virus indicated that they have molecular masses of 39·5 kDa and 40 kDa (respectively), whereas estimates from deduced amino acid sequences suggested that they have molecular masses of 32·8 kDa and 34·1 kDa. Comparison of the NIb and coat protein sequences with other viruses showed that NLV and MacMV have close affinities with viruses of the Potyviridae and suggests that they should form a new genus of the family.
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A nonoccluded reovirus of the olive fly, Dacus oleae.
More LessWe have isolated paraspherical viral particles, 60 nm in diameter, from adults of the olive fly (Dacus oleae) collected in Greece. The virus actively replicated in midgut epithelial cells and in advanced infections virions accumulated in microvilli. They were released in the gut lumen and were very abundant in fly faeces. The virions exhibited the salient features of reoviruses, with an external shell and an internal core with a tubular subunit protruding at each vertex of the icosahedron. The viral genome consisted of ten segments of doublestranded RNA totalling 23·4 kbp. Based on its overall properties, this virus can be considered as a nonoccluded insect reovirus.
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A physical map of the Mamestra configurata nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene.
More LessThe genome structure of a nucleopolyhedrovirus (NPV) isolated from the bertha armyworm, Mamestra configurata Walker (Lepidoptera: Noctuidae) (MacoNPV) was analysed with six restriction endo-nucleases (REN): Bam HI, Eco RI, Hin dIII, Pst I, Sma I, and Xho I. More than 70 MacoNPV REN fragments were cloned into plasmids pUC18 and pBluescript SK( + ). The physical map with 112 restriction sites for the above REN was constructed using double digests and Southern blot hybridization analysis of the MacoNPV DNA clones. The size of the DNA genome of the MacoNPV-90/2 isolate used for this study was estimated at 156 kbp based on REN fragment sizes. The position of the polyhedrin gene, which has by convention been used as the zero point of the REN maps of NPV, was determined by hybridizing the Autographa californica multicapsid nucleopolyhedrovirus Hin dIII-V fragment clone, which contains most of the polyhedrin gene, with genomic blots of MacoNPV. The cloned MacoNPV fragments identified as containing the polyhedrin gene were sequenced and an ORF coding for a 246 amino acid polypeptide with 98·7% sequence identity with Panolis flammea nucleopolyhedrovirus (PaflNPV) polyhedrin protein was identified. The putative polyhedrin gene sequence had 97·2% and 91·2% identity with the PaflNPV and Mamestra brassicae multicapsid nucleopolyhedrovirus polyhedrin gene sequences, respectively, and also contained an upstream region identical to the highly conserved 12 bp consensus sequence TGTAAGTAATTT typical of NPV very late genes.
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Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.
Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons: (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by antisense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the human c-myc gene was inserted downstream from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus and tested in bioassays on Spodoptera frugiperda larvae. Western blot analysis with a human c-myc antibody revealed an endogenous protein band which bound specifically to these antibodies. This band disappeared more rapidly from cells infected with the antisense c-myc recombinant virus than from those infected with c-myc-negative virus. Results of bioassays showed that the antisense construct stopped feeding as soon as the polyhedrin promoter-driven transcripts accumulated, followed shortly by death of the larvae. These results suggest that c-myc-like protein(s) exist in insects and that the antisense strategy is an effective approach to virus insecticide production.
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Scrapie strains retain their distinctive characteristics following passages of homogenates from different brain regions and spleen.
More LessThe molecular basis of differences among scrapie strains is unknown. The prion theory posits that there are differences in the conformation of the host protease-resistant protein (PrP) molecules and that these differences are responsible for scrapie strains. A corollary of this theory is that the origin of host PrP variation resides in different neuronal cell types. To assess this concept, preparations from three brain regions (cerebrum, cerebellum and olfactory bulb) and from spleen were passaged in C57BL mice by intracerebral injection. After three passages of three scrapie strains in this manner, homogenates of each brain region and spleen were tested for several of the characteristics that distinguish the three strains: (1) the rank order of incubation periods in C57BL mice, (2) induction of obesity in SJL mice and (3) comparative incubation periods in mice with three genotypes for the scrapie incubation period marker. Analysis revealed that virtually all of the criteria that distinguished the three strains prior to passages of the three brain regions and spleen were retained after this series of passages. This finding argues against cellular-based PrP differences providing a basis for strain specificity.
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