- Volume 82, Issue 3, 2001
Volume 82, Issue 3, 2001
- Animal: DNA Viruses
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Divergent replication kinetics of two phenotypically different parvoviruses of rats
More LessRat virus (RV) is an important infectious agent of laboratory rats because of its high prevalence and capacity to disrupt research. Additionally, RV infection serves as a model for characterizing virus–host interactions during acute, persistent and prenatal infection. Our research has examined the pathogenesis of two RV strains, RV-UMass and RV-Y. RV-UMass is more pathogenic, causes a higher level of persistent infection and transmits to the foetus after oronasal inoculation of the pregnant dam. To determine in vitro distinctions between the strains that may account for these differences and to provide a benchmark for characterizing virus replication in vivo, synchronized in vitro replication of both RV strains was defined and compared. The results demonstrated that RV replication has replicative intermediates, virus transcripts and proteins similar to those reported for the prototype parvovirus, minute virus of mice. However, the replicative cycle of RV-UMass was 12 h compared with 24 h for RV-Y, and RV-UMass and RV-Y differed in kinetics of virus DNA replication, transcription and protein accumulation. Additionally, in situ analysis correlated well with kinetics data as determined by Southern and Northern blot analysis. Sequence comparisons between the strains also determined coding differences that may contribute to phenotypic differences.
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Recombination, pseudorecombination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda
More LessThe molecular variability of cassava geminiviruses occurring in Uganda was investigated in this study. Infected cassava plants and whiteflies were collected from cassava plantings in different geographical areas of the country and PCR was used for molecular characterization of the viruses. Two complete sequences of DNA-A and -B from African cassava mosaic virus (ACMV), two DNA-A sequences from East African cassava mosaic virus (EACMV), two DNA-B sequences of EACMV and the partial DNA-A nucleotide sequence of a new virus strain isolated in Uganda, EACMV-UG3, are reported here. Analysis of naturally infected cassava plants showed various assortments of DNA-A and DNA-B of the Ugandan viruses, suggesting the occurrence of natural inter- and intraspecies pseudorecombinations and a pattern of cassava mosaic disease (CMD) more complex than previously reported. EACMV-UG2 DNA-A, which contains a recombinant fragment between ACMV and EACMV-UG1 in the coat protein gene that resembles virus from Tanzania, was widespread in the country and always associated with EACMV-UG3 DNA-B, which probably resulted from another natural recombination event. Mixed infections of ACMV-UG and EACMV-UG in cassava and whiteflies were detected in most of the regions where both viruses occurred. These mixed-infected samples always showed extremely severe CMD symptoms, suggesting a synergistic interaction between ACMV-UG and EACMV-UG2. The first demonstration is provided of infectivity of EACMV clones to cassava, proving conclusively that the pseudorecombinant EACMV-UG2 DNA-A+EACMV-UG3 DNA-B is a causal agent of CMD in Uganda.
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Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus
More LessIn the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.
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The size of encapsidated single-stranded DNA determines the multiplicity of African cassava mosaic virus particles
More LessTransgenic Nicotiana benthamiana plants harbouring a defective interfering (DI) DNA of African cassava mosaic virus (ACMV) and control plants were inoculated with ACMV. Virus particles were purified from infected plants, separated in sucrose gradients and fractions were analysed by Southern blotting. Transgenic plant-derived virus particles taken from the top fractions of sucrose gradients contained DI DNA, middle fractions contained a mixture of genomic and DI DNA and bottom fractions contained a mixture of multimeric, genomic and DI DNA. Virus particles from selected top, middle and bottom fractions were analysed by electron microscopy. In fractions containing only DI DNA, isometric particles of 18–20 nm were detected. In fractions containing DI DNA as well as genomic size DNA, isometric and geminate particles were found. Fractions containing multimeric size DNA were found to comprise particles consisting of three subunits adjacent to geminate particles. From these data, it is concluded that the size of encapsidated DNA determines the multiplicity of ACMV particles.
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Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system
More LessPotyvirus CI RNA helicase is a protein involved in RNA genome replication and virus movement. The protein aggregates in the cytoplasm of infected cells to form typical cylindrical inclusions. A yeast two-hybrid system was used to analyse interactions of the CI RNA helicase from plum pox potyvirus (PPV) with itself and with other viral proteins. No interactions could be detected of full-length CI protein with itself or with PPV P3/6K1, NIa, NIb or CP proteins. However, positive self-interactions were detected for N-terminal fragments of the CI protein, allowing the mapping of a CI–CI binding domain to the N-terminal 177 aa of the protein. Further deletion analysis suggested that several regions of this domain contribute to the interaction. Moreover, pull-down experiments demonstrate that, at least in vitro, full-length PPV CI protein is able to self-interact in the absence of other virus or plant factors.
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The DNA form of a retroviroid-like element characterized in cultivated carnation species
More LessCarnation small viroid-like RNA (CarSV RNA) is a small (275 nt), circular molecule which is unique among plant viroid-like RNAs in having a tandemly repeated homologous DNA. This DNA form was found fused to DNA sequences of carnation etched ring caulimovirus (CERV) in certain Spanish carnation plants. The observation of a growth abnormality consisting of extensive shoot proliferation in cultivated carnations in Hungary prompted the molecular analysis of these plants, in which both CarSV RNA and DNA forms were detected. Several CarSV DNA sequences were characterized in various Dianthus caryophyllus cultivars which were symptomless or showed different symptoms. CarSV DNA forms showing minor sequence heterogeneities and deletions occurred in the same plant. Unit-length CarSV DNA sequences were proven to accumulate in the plant cell nucleus. The plants studied here were not infected by any of the viruses (including CERV) or other cellular pathogens described previously in carnation.
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