Engineering cowpea mosaic virus RNA-2 into a vector to express heterologous proteins in plants.
A series of new cowpea mosaic virus (CPMV) RNA-2-based expression vectors were designed. The jellyfish green fluorescent protein (GFP) was introduced between the movement protein (MP) and the large (L) coat protein or downstream of the small (S) coat protein. Release of the GFP inserted between the MP and L proteins was achieved by creating artificial processing sites each side of the insert, either by duplicating the MP-L cleavage site or by introducing a sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. Eight amino acids derived from the C-terminus of the MP and 14-19 amino acids from the N-terminus of the L coat protein were necessary for efficient processing of the artificial Gln/Met sites. Insertion of the FMDV 2A sequence at the C-terminus of the GFP resulted in a genetically stable construct, which produced particles containing about 10 GFP-2A-L fusion proteins. Immunocapture experiments indicated that some of the GFP is present on the virion surface. Direct fusion of GFP to the C-terminus of the S coat protein resulted in a virus which was barely viable. However, when the sequence of GFP was linked to the C-terminus by an active FMDV 2A sequence, a highly infectious construct was obtained.
Gopinath
K
K
Laboratory of Molecular Biology, Agricultural University, Dreijenlaan 3, Wageningen, 6703 HA, The Netherlands.
Wellink
J
J
Porta
C
C
Taylor
K M
KM
Lomonossoff
G P
GP
van Kammen
A
A
D000595:Amino Acid Sequence; D005537:Aphthovirus / Q000235:genetics; D002213:Capsid / Q000235:genetics; D002384:Catalysis; D017798:Comovirus / Q000235:genetics* / Q000648:ultrastructure; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D053830:Plant Viral Movement Proteins; D010944:Plants / Q000235:genetics* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D014771:Virion / Q000235:genetics / Q000648:ultrastructure
D008164:Luminescent Proteins; D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins