- Volume 79, Issue 9, 1998
Volume 79, Issue 9, 1998
- Articles
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Induction of neutralizing antibodies by synthetic peptides representing the C terminus of coxsackievirus A9 capsid protein VP1
More LessThe arginine-glycine-aspartic acid motif at the C terminus of coxsackievirus A9 capsid protein VP1 has been shown to play a role in specific attachment of the virus to α v β 3 integrin on the host cell surface. The C-terminal region of the VP1 protein has also been shown to be highly antigenic by using peptide scanning techniques. To find out whether this region contains a neutralizing epitope, three overlapping peptides covering the C-terminal end of VP1 were synthesized and rabbit antisera were raised against these peptides. Neutralization of the virus was observed with all three antipeptide antisera in A549 cells and with two antisera in RD cells.
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Antiviral activity of bovine collectins against rotaviruses
More LessThe antiviral activity against rotaviruses of three bovine collectins, conglutinin, collectin-43 (CL-43) and bovine SP-D, was examined. As shown by ELISA and Western blot, all three collectins bound to the Nebraska calf diarrhoea virus bovine strain of rotavirus, and specifically to the VP7 glycoprotein. Inhibition by mannose or EDTA confirmed that binding was mediated through the lectin domains of the collectins. Binding resulted in haemagglutination inhibition and neutralization of rotavirus in-fectivity, CL-43 displaying the highest activity in both types of assay. In contrast, conglutinin was the most potent of the three collectins against influenza virus A/HKx31. Neutralization of rotaviruses by the lectins was dependent on glycosylation of VP7. Furthermore, rotaviruses adapted to growth in Madin-Darby bovine kidney cells, and thus bearing carbohydrate of bovine origin, remained sensitive to neutralization, although slightly less so than virus stocks propagated in the monkey kidney cell line MA104. These findings provide the first description of antiviral activity of collectins against a non-enveloped virus and may indicate a potential role for collectins in host defence against bovine rotavirus infection.
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Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants
More LessBean yellow dwarf virus (BeYDV) is an atypical member of the geminivirus genus Mastrevirus that infects dicotyledonous plants. BeYDV DNA contains six open reading frames (ORFs) with the capacity to encode proteins in excess of 10 kDa. Two virion- sense ORFs (V1 and V2) and two complementary- sense ORFs (C1 and C2) have homologues in all mastreviruses, while ORFs C3 and C4 are not conserved. To investigate their functions, each of the ORFs has been truncated by either frame- shifting or the introduction of a stop codon. We demonstrate that an ORF V1 mutant replicated efficiently in Nicotiana tabacum protoplasts but was unable to systemically infect Phaseolus vulgaris and Datura stramonium, consistent with a role for V1 protein in virus movement. However, the mutant was able to systemically infect Nicotiana benthamiana although the onset of symptoms was appreciably delayed in comparison with wild-type virus. Disruption of ORF V2, encoding the coat protein, prevented systemic infection of all three hosts but the mutant replicated in protoplasts. Both ORF C1 and ORF C2 were essential for replication in protoplasts. Modification of the complementary- sense splice donor and acceptor sequences also prevented replication. Removal of the intron prevented systemic infection, although the intronless mutant was able to produce functional replication- associated protein (Rep) and replicated efficiently in protoplasts. ORFs C3 and C4 were not required for systemic infection. Our results indicate that four ORFs are spatially and functionally conserved in mastreviruses that infect both monocotyledonous and dicotyledonous plants.
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cDNA cloning and molecular characterization of cherry green ring mottle virus
More LessThe complete nucleotide sequence of the cherry green ring mottle virus (CGRMV) genome was determined to be 8372 nt excluding a 3′ poly(A) tail. Based on computer analysis and sequence comparison, five open reading frames (ORFs) were identified on the virion strand encoding: a putative RNA-dependent RNA polymerase, a triple gene block and a coat protein. Two other ORFs with M r values over 10000 and internal to the helicase and coat protein genes, but of unknown function, were also identified. Sequence and genome structure comparisons with other filamentous viruses indicated that CGRMV is most similar to apple stem pitting virus, some carlaviruses and potexviruses. However, it is different from members of any of these virus groups in regard to sequence homology and genome organization. A chimeric fusion coat protein was expressed in E. coli and antibodies specific for the CGRMV coat protein were raised in rabbits. The antibody was used in Western blot analyses to detect the CGRMV coat protein in infected cherry tissue.
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Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes
More LessA 3·2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66·7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopoly- hedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system.
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Apoptosis resulting from superinfection of Heliothis zea virus 1 is inhibited by p35 and is not required for virus interference
More LessSuperinfection of Spodoptera frugiperda insect cells that are persistently infected with Heliothis zea 1 (Hz-1) virus induces general cellular apoptosis and subsequently results in homologous virus interference. Since apoptosis correlates closely with both a significant decrease in yield of virus progeny and expansion of virus infection among cells, further experiments were designed to verify the direct association of apoptosis with homologous interference. It was found that superinfection-induced apoptosis can be efficiently blocked by the stable transfection of p35 into cells before or after the establishment of persistent virus infection. However, persistently infected cells are still strongly resistant to the challenge of Hz-1 virus, indicating that the induction of apoptosis is not essential for the resulting homologous Hz-1 virus interference. Replication and transcription of viral genomes are greatly retarded upon Hz-1 virus superinfection of persistently infected cells, whether stably transfected with p35 or not, suggesting that upon superinfection, the decreasing yield of virus progeny in these persistently infected cells is caused by a blockage early after virus infection.
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