Subcellular distribution analysis of the cucumber mosaic virus 2b protein

The cucumoviral 2b protein is a viral counterdefence factor that interferes with the establishment of virus-induced gene silencing in plants. Syntheti c peptides were used to generate an antibody to the 2b protein encoded by t he Fny strain of cucumber mosaic virus (Fny-CMV). This polyclonal antibody was able to recognize the Fny-CMV 2b protein in a 10 000 g pellet fraction of infected tobacco. No protein of equivalent size was detected in mock-ino culated or tobacco mosaic virus-infected samples. This represents the first demonstration of 2b protein expression by a subgroup I strain of CMV. Subc ellular fractionation experiments on CMV-infected tobacco leaf tissue showe d that the Fny-CMV 2b protein accumulated within a fraction that sedimented at forces of less than 5000 g and that the 2b protein was solubilized only by treatment with urea or SDS. These results suggested that the 2b protein associates either with the nucleus or cytoskeleton of the host cell. Furth er analysis showed that the 2b protein was enriched in a fraction that sedi mented through a 2.2 M sucrose cushion. This fraction was also enriched in histones, suggesting that the CMV 2b protein associates preferentially with the host cell nucleus.