The Abutilon mosaic virus (AbMV, Geminiviridae) DNA B component encodes a movement protein (MP), which facilitates viral transport within plants and affects pathogenicity. The presence of phosphorylated serine and threonine residues was confirmed for MP expressed in yeast and Nicotiana benthamiana by comparative Western blot analysis using phospho-amino acid- and MP-specific immunodetection. Mass spectrometry of yeast-derived MP identified three phosphorylation sites located in the C-terminal domain (Thr-221, Ser-223 and Ser-250). To assess their functional relevance in plants, several point mutations were generated in the MP gene of DNA B, which replace Thr-221, Ser-223 and Ser-250, either singly or in combinations, with either an uncharged alanine or a phosphorylation-mimicking aspartate residue. When co-inoculated with DNA A, all mutants were infectious. In systemically infected plants the symptoms and/or viral DNA accumulation were significantly altered for several of the mutants.

The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.

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The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. IMPORTANCEThe (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology.

Geminiviral minichromosomes were purified to explore epigenetic modifications. The levels of methylation in their covalently closed circular DNA were examined with the help of methylation-dependent restriction (MdR). DNA with 12 superhelical turns was preferentially modified, indicating minichromosomes with 12 nucleosomes leaving an open gap. MdR digestion yielded a specific product of genomic length, which was cloned and Sanger-sequenced, or amplified following ligation-mediated rolling circle amplification and deep-sequenced (circomics). The conventional approach revealed a single cleavage product indicating specific methylations at the borders of the common region. The circomics approach identified considerably more MdR sites in a preferential distance to each other of ~200 nts, which is the DNA length in a nucleosome. They accumulated in regions of nucleosome-free gaps, but scattered also along the genomic components. These results may hint at a function in specific gene regulation, as well as in virus resistance.