Hipp K, Schäfer B, Kepp G, Jeske H.

Viruse, 2016 Jul 8;8(7). pii: E190. doi: 10.3390/v8070190.

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.

See: http://www.mdpi.com/1999-4915/8/7/190/htm

Figure 4. Interaction of CP with ssDNA. Extract supernatants from CP-expressing (CP) or control cells (V) were incubated with ssDNA from pCLV1.3A (CLV), pBlue:ACMVA(v) (A(v)), or pBlue:ACMVA(c) (A(c)). Complexes or ssDNA alone were precipitated with polyethylene glycol and analyzed on an ethidium bromide stained 0.7% agarose gel (a) or, after blotting onto nylon (b) or nitrocellulose (c) membranes, for viral DNA or CP detection using hybridization with an ACMV DNA A-specific probe (b) or an CP-specific antibody (c), respectively. The corresponding positions of complexes with ssDNA and CP are indicated by asterisks, the positions of ssDNA by arrowheads. M: PstI fragments of λ DNA with bp indicated. Hybridization standards: 100 and 10 pg of linearized full-length ACMV DNA A (10, 100). Bands in (a): rRNA.