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Volume 76,
Issue 3,
1995
Volume 76, Issue 3, 1995
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Helper virus-free transfer of human immunodeficiency virus type 1 vectors
More LessRecombinant vectors based on the type 1 human immunodeficiency virus (HIV-1) can be used to deliver genes into cells expressing the HIV receptor, CD4. We have used a transient RNA packaging system to compare the safety and efficacy of HIV-1 vector transduction by wild-type and replication-deficient helper viruses. Helper virus-free vector transfer was consistently achieved when the helper virus gag-pol and env genes were expressed from separate plasmids such that two recombination events were required to form an infectious genome. Other forms of attenuation, such as deletion of the 5′ ψ region, were inadequate to prevent helper virus transmission. Vector transduction by the wild-type and non-replicating helper viruses occurred with comparable efficiency except in instances where efficient vector RNA expression was dependent upon transactivating factors supplied by the helper virus. These data demonstrate the feasibility of safe gene transfer using HIV-1 vectors.
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Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein genes similar to 1975 human strains
Of the four pandemic strains of human influenza A virus observed this century, the 1977 virus strain was very similar in all genes to a 1950 isolate. Since mammalian influenza A viruses change annually by genetic drift, this reappearance could only be attributed at that time to conservation of the virus in a frozen state. We report here the isolation of swine influenza A viruses with haemagglutinin and nucleoprotein genes which are virtually identical to those of the human virus that circulated in 1975. We have also found serological evidence that this virus is circulating extensively in Quebec swine herds. We propose that human-like H3N2 influenza A strains may remain invariant for long periods in swine, which may serve as a reservoir for human pandemics.
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Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein
More LessThe fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One singlesite mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.
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Diversity of genotypes of hepatitis C virus in southern India
More LessA second generation assay for antibody to hepatitis C virus (anti-HCV) was used to screen 78 southern Indian individuals with a high risk of infection. RT-PCR targeted at the 5′ end untranslated region (5′UTR) of the HCV genome was used to evaluate evidence of viraemia in 32 anti-HCV positive sera. The PCR products amplified from the 5′UTR of the HCV genome from 24 patients were sequenced, revealing the existence of two distinct groups of sequences: 21 corresponded to HCV type 1 while the other three sequences had 95% to 99% identity to HCV type 3. Two of these three isolates had more than 90% nucleotide identity in the NS5 region to established 3b sequences whereas the other had less than 74% nucleotide identity to any of the published genotype 3 (3a, 3b, 3c, 3d, 3e and 3f) sequences. However, a search of the EMBL nucleotide database revealed 91% identity to the unpublished sequence of an isolate of HCV from Indonesia. We provide evidence that these two isolates may represent a novel subtype within genotype 3. Our data also suggest that HCV genotype 1 predominates over HCV genotype 3 in southern India.
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Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein
More LessThree potential AG*X cleavage sites have previously been identified in vaccinia virus (VV) P4a precursor, namely AG*N, AG*S and AG*T. Utilization of the COOH-proximal AG*T site in P4a leads to release of a 23 kDa product (‘23K’). Here we propose that cleavage at the AG*S site alone is responsible for release of 4a, based on peptide mapping and microsequencing which demonstrated that the NH2 terminus of 4a is co-terminal with P4a, thus indicating that the AG*N site is excluded from proteolytic processing. Proteolysis of P4a at AG*S and AG*T to yield 4a and 23K should theoretically also liberate an intervening 9 kDa peptide (‘9K’), although efforts to isolate this peptide have been repeatedly unsuccessful. To investigate the fate of this intervening peptide, mutation of the P4a coding sequence at the AG*S or AG*T site, followed by transient expression in VV-infected cells, lead to the synthesis of unique 4a-9K or 9K–23K chimeric protein products. This implies that neither end of the intervening 9K peptide is intrinsically destabilizing, and that its supposed degradation may be suppressed when it remains associated with 4a or 23K.
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In situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus RNA 3
More LessThe in situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus (BNYVV) RNA 3 and of the BNYVV coat protein (CP) was studied by immunoelectron microscopy in infected leaf and root cells of Chenopodium murale and C. quinoa. The CP was detected in the cytoplasm of all cell types except xylem, sieve elements, and companion cells. P25 was detected in the cytoplasm and nuclei of the same cell types. The intensity of CP labelling varied depending upon the stage of infection of the cell, whereas the P25 labelling intensity was similar in newly infected cells and in cells at later stages of infection. These results suggest that P25 may be synthesized at an earlier stage of infection than CP. Its presence in the nuclei of newly infected cells may be related to the reported effect of P25 on leaf symptom development.
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Populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic RNA molecules
More LessPlants infected by citrus tristeza virus (CTV) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. The CTV particles and RNA extracts from purified CTV preparations were fractionated by sucrose gradient centrifugation and the RNA molecules from different fractions translated in a reticulocyte translation system. Fractions containing predominantly an RNA band of approximately 3.2 kb directed the synthesis of CTV coat protein (CP), which in SDS-PAGE had an estimated molecular mass of 28 kDa. Three additional polypeptides, with estimated sizes of 21 kDa, 23 kDa and 27 kDa, were translated from a range of RNA molecules smaller than 3.2 kb. Hybridization with cDNA to the CP gene (CTV-CPG) and with a 350 base clone complementary to the 3′ and 5′ termini of the genes for CTV p20 and p23.5, respectively, indicated that preparations of CTV particles contain, in addition to the genomic (20 kb) RNA, two sub-genomic RNA molecules of 3.2 kb and 2.4 kb and probably also two smaller molecules of 1.6 kb and 0.9 kb. Only the 3.2 kb RNA, its corresponding dsRNA molecule and populations of larger RNAs, including the 20 kb genomic RNA, hybridized with a CTV-CPG probe, thus conflicting with our previous assignment of the CTV-CPG to the 0.8 kbp dsRNA. Based on these results we propose that distinct populations of CTV particles encapsidate smaller RNAs which were formed as a nested set of subgenomic RNAs. Sequence analysis of 2540 nucleotides downstream to CTV-CPG of strain VT revealed four open reading frames (ORFs) potentially encoding, in the 5′ to 3′ direction, 18 kDa (p18), 13 kDa (p13), 20 kDa (p20) and 23.5 kDa (p23.5) proteins. The CTV-VT ORFs showed variable but usually close levels of homology with the corresponding ORFs of CTV-T36 from Florida.
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