In vivo and in vitro characterization of the movement protein of cucumber mosaic virus.

摘要

Cucumber mosaic virus (CMV) is a tripartite single-stranded (ss) RNA virus with a wide host range. Although it has been well studied genetically, knowledge about the cell-to-cell movement of CMV is relatively limited. This study was conducted to characterize the putative movement protein (3a) of CMV both in vivo and in vitro in an attempt to elucidate the mechanism of the cell-to-cell movement of CMV. The 3a gene of CMV was over-expressed in E. coli cells and a polyclonal antibody was produced against the E. coli-expressed CMV 3a protein. Examination of the subcellular distribution of the CMV 3a protein by immunological detection revealed that most of the 3a protein was present in a soluble form in the cytoplasmic fraction in the young leaves of CMV-infected tobacco or the 3a-expressing transgenic tobacco plants. However, in either older infected tobacco leaves or full-developed transgenic tobacco leaves, most of the 3a was found in the cell wall-enriched fraction in predominantly a degraded form. The expressed 3a protein isolated from E. coli also showed the ability to bind ss nucleic acid and GTP in vitro. Alanine-scanning mutations were generated in the CMV 3a protein to study their effects on the in vitro RNA and GTP binding activities as well as the biological functions of the 3a protein. While four of the nine mutants, with changes in the middle portion of the 3a protein rendered the protein defective in movement, the other five mutants, with changes in the N-terminal one-third of the 3a protein did not affect the movement of the virus, but had some effects on the symptoms shown on some plant hosts. The mutant 3a proteins showed different levels of in vitro RNA binding activity, ranging from 5% to 130% of the level by the wild type 3a protein. All mutant 3a proteins were able to bind GTP. Thus, no strict correlation could be shown between the biological functions and the RNA or GTP binding activity of the 3a protein.; Finally, the CMV 3a protein was isolated from CMV-infected tobacco plants using the hexa-histidine tagging strategy. The His-tagged 3a protein isolated from plants demonstrated the ability to bind ss RNA like the one isolated from E. coli in UV-crosslinking assays. Notably, some host proteins could be co-purified with the His-tagged 3a protein from CMV-infected plants. Further characterization of these co-purified proteins may give valuable information about the virus-host interactions in the process of cell-to-cell movement.