Abstract
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.
Highlights
African cassava mosaic virus (ACMV) is a small plant pathogenic virus belonging to the Begomovirus genus of the Geminiviridae family [1]
The ACMV AV1 ORF was transferred into a fission yeast plasmid for expression under the supernatant (Figure 1a, arrowheads) as well as in the pellet fractions (Figure 1b, arrowheads) after inducible nmt1 promoter [43]
The successful expression of ACMV capsid proteins (CPs) in fission yeast extends the list of several geminiviral proteins that were produced in this organism [35,36,46,47,48,49]
Summary
African cassava mosaic virus (ACMV) is a small plant pathogenic virus belonging to the Begomovirus genus of the Geminiviridae family [1]. It is one of the most prevalent pathogens of cassava in Africa [2,3,4]. Several geminiviral CPs have been examined by in vitro binding studies after bacterial expression. Three regions of ACMV CP—at the N-terminus, within the central part, and the C-terminus—contain nuclear localization signals (NLS) [28]. CP was expressed in fission yeast under the control of an inducible promoter to study its’ in vitro assembly with ssDNA
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