DNA Methylation Suppression by Bhendi Yellow Vein Mosaic Virus (original) (raw)
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Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms
Nucleic Acids Research, 1993
Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells.
Viral genome methylation as an epigenetic defense against geminiviruses
Journal of virology, 2008
Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through doublestranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2 ؊ mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.
Progression of geminivirus-induced transgene silencing is associated with transgene methylation
New Phytologist, 2002
• The association of viral-induced gene silencing (VIGS) elicited by a DNA virus with DNA methylation of the silenced transgene was studied. • 35S-Green fluorescent protein (GFP) transgenic Nicotiana benthamiana were treated with an inhibitor of DNA methylation, 5-azacytidine (5-Aza-C), and VIGS of the transgene was observed upon inoculation with tomato golden mosaic virus carrying the GFP coding sequence. • The onset of VIGS of the 35S-GFP transgene occurred 14-16 d after inoculation in both control and 5-Aza-C-treated plants. At this stage, the silencing was observed in localized regions. Silencing was uniform by 30 d after inoculation in plants that had methylated GFP-DNA, whereas plants that continued to display the same phenotype as seen at 14-15 d after inoculation had hypomethylated GFP-DNA. Viral expression of GFP persisted in pockets throughout the life of infected plants. • This is the first demonstration of a correlation between post transcriptional gene silencing induced by a DNA virus, and transgene methylation. The results suggest that, while DNA methylation is not necessary for the initiation of silencing, the progression of silencing is affected by inhibition of DNA methylation.
Scientific Reports, 2016
DNA methylation and post-transcriptional gene silencing play critical roles in controlling infection of single-stranded (ss) DNA geminiviruses and ssRNA viroids, respectively, but both pathogens can counteract these host defense mechanisms and promote their infectivity. Moreover, a specific role of DNA methylation in viroid-host interactions is not yet confirmed. Here, using an experimental system where two nuclear-replicating agents, the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSV) and potato spindle tuber viroid (PSTVd), co-infect their common host tomato, we observed that PSTVd severely interferes with TYLCSV infectivity and accumulation, most likely as a consequence of strong activation of host DNA methylation pathways. In fact, PSTVd alone or in co-infection with TYLCSV significantly upregulates the expression of key genes governing DNA methylation in plants. Using methylation-sensitive restriction and bisulfite conversion assays, we further showed that PSTVd inf...
Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression
Proceedings of the National Academy of Sciences, 1983
The effect of DNA methylation on polyoma virus and simian virus 40 gene expression was investigated. For this purpose, the cytosines of all C-G dinucleotides of the viral DNAs were methylated by the use of rat liver methylase and the completeness of methylation was verified by dinucleotide analysis and restriction endonuclease treatment. The biological activity of unmethylated and fully methylated DNAs was tested by microinjecting them into tissue culture cells. The functions analyzed included early and late viral gene expression, viral DNA replication, oncogenic transformation efficiency, and virus maturation. No difference in any of these biological functions was observed between methylated and unmethylated DNA. Early gene expression of methylated DNA is not the result of demethylation because viral DNA reextracted from the injected cells, under nonpermissive conditions, retained the methylation pattern of the input DNA. In contrast, viral DNA extracted from transformed cells or from intact virus particles was partially or completely demethylated.
J Gen Virol, 2009
DNA b is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA b contains a single gene, bC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the b-glucuronidase reporter gene driven by a bC1 promoter-deletion series of the DNA b associated with cotton leaf curl Multan virus identified a 68 nt region (between "139 and "207) which is important for bC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the bC1 start codon. Mutation of the G-box resulted in a significant reduction in bC1 promoter activity and DNA b replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA b.
Coat protein gene of an isolate of Bhendi yellow vein mosaic virus (BYVMV) and the associated DNA-beta satellite obtained from eastern India were cloned and nucleotide sequences were analyzed to know its molecular status and phylogenetic relationship. Multiple nuclear localization signals (NLSs) were found at the N-terminal region of coat protein and endoplasmic reticulum membrane retention signal in βC1 protein of beta satellite. The sequence of coat protein gene shared highest sequence identity, with that of the south Indian isolate (BYVMV-[Mad]:Var3, Accession no. AJ278861) and formed cluster with it, whereas the beta satellite molecule (BYVB-[IN:Bar:06]), characterized in the study, shared highest sequence identity and formed cluster with that recorded to be associated with another isolate of BYVMV on bhendi plants from Pakistan (BYVB-[PK:Bah:97]) (Accession No. AJ316030). . 2008. Molecular characterization and computational analysis of coat protein gene of an east Indian isolate of Bhendi yellow vein mosaic virus and associated DNA-beta satellite.
Journal of General Virology, 2009
DNA b is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA b contains a single gene, bC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the b-glucuronidase reporter gene driven by a bC1 promoter-deletion series of the DNA b associated with cotton leaf curl Multan virus identified a 68 nt region (between "139 and "207) which is important for bC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the bC1 start codon. Mutation of the G-box resulted in a significant reduction in bC1 promoter activity and DNA b replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA b.
Proceedings of the National Academy of Sciences, 1981
The Mov-3 substrain of mice carries Moloney murine leukemia virus as a Mendelian gene in its germ line. All mice segregating the Mov-3 locus activate virus and develop viremia and leukemia. The integrated provirus (i.e., Mov-3 locus) was molecularly cloned from Mov-3 liver DNA as a 16.8 kilobase longEcoRI fragment. Comparison ofthe cloned and genomic Mov-3 specific EcoRI fragment by restriction enzyme analysis showed no differences in the size ofthe fragments, indicating that no major sequence rearrangements occurred during cloning. The genomic and cloned Mov-3 DNAs were compared for methylation and infectivity. Analysis with Hha I showed that the genomic proviral and the flanking mouse sequences were methylated at cytosine residues, in contrast to the cloned Mov-3 locus. The cloned Mov-3 locus, however, was highly infectious in a transfection assay (1 x 10-5 plaque-forming unit per viral genome) in contrast to the genomic Mov-3 DNA (<10 per viral genome). Our results suggest that genes containing 5-methylcytosine are not expressed after transfection into susceptible cells and that removal of the methyl groups by molecular cloning in prokaryotes leads to expression generating infectious proviral DNA. If gene expression of transfected DNA is controlled by mechanisms that are relevant for gene expression in the animal, this suggests that DNA methylation may play a causative role in eukaryotic gene regulation.