Suppression of methylation-mediated transcriptional gene silencing by ßC1-SAHH protein interaction during geminivirus-betasatellite infection.

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA ß. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, ßC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2⁻ mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or ßC1 expression. We also demonstrate that while TYLCCNB or ßC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that ßC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that ßC1 protein inhibits SAHH activity in vitro. That ßC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by ßC1 stabilizes geminivirus/betasatellite complexes.

RNA silencing directed against geminiviruses: post-transcriptional and epigenetic components.

It is well-established that plants use cytoplasmic, post-transcriptional gene silencing (PTGS) as a defense against RNA viruses and DNA virus transcripts. More recently, it has become clear that small RNA-directed methylation leading to transcriptional gene silencing (TGS) is also used as a defense against DNA virus chromatin. Here we use the DNA-containing geminiviruses as models to discuss what is currently known about both types of antiviral silencing, and viral suppression of PTGS and TGS as a counterdefense.

Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.