title: Studies on the movement of cowpea mosaic virus using the jellyfish green fluorescent protein.
abstract: The jellyfish green fluorescent protein (GFP) coding sequence was used to replace the coat protein (CP) genes in a full-length cDNA clone of CPMV RNA-2. Transcripts of this construct were replicated in the presence of RNA-1 in cowpea protoplasts, and GFP expression could be readily detected by fluorescent microscopy. It was not possible to infect cowpea plants with these transcripts, but combined with a mutant RNA-2, in which the 48-kDa movement protein (MP) gene has been deleted infection did occur. With this tripartite virus (CPMV-TRI) green fluorescent spots were visible under UV light on the inoculated leaf after 3 days and a few days later on the higher leaves. These results show that the polyproteins encoded by RNA-2 do not possess an essential function in the virus infection cycle and that there is, contrary to what we have found so far for the proteins encoded by RNA-1, no need for a tight regulation of the amounts of MP and CPs produced in a cell. Subsequently, the GFP gene was introduced between the MP and CP genes of RNA-2 utilizing artificial proteolytic processing sites for the viral proteinase. This CPMV-GFP was highly infectious on cowpea plants and the green fluorescent spots that developed on the inoculated leaves were larger and brighter than those produced by CPMV-TRI described above. When cowpea plants were inoculated with CPMV RNA-1 and RNA-2 mutants containing the GFP gene but lacking the CP or MP genes, only single fluorescent epidermal cells were detected between 2 and 6 days postinoculation. This experiment clearly shows that both the capsid proteins and the MP are absolutely required for cell-to-cell movement.
authors.affiliation: Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Verver
authors.forename: J
authors.initials: J
authors.lastname: Wellink
authors.forename: J
authors.initials: J
authors.lastname: Van Lent
authors.forename: K
authors.initials: K
authors.lastname: Gopinath
authors.forename: A
authors.initials: A
authors.lastname: Van Kammen
mesh_terms: D000818:Animals; D002213:Capsid / Q000235:genetics; D003001:Cloning, Molecular; D017798:Comovirus / Q000235:genetics / Q000502:physiology*; D007887:Fabaceae / Q000821:virology; D017353:Gene Deletion; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009068:Movement; D053830:Plant Viral Movement Proteins; D010946:Plants, Medicinal; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D037401:Scyphozoa; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins