In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA.
The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.
Zhao
X
X
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Fox
J M
JM
Olson
N H
NH
Baker
T S
TS
Young
M J
MJ
D001483:Base Sequence; D017795:Bromovirus / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000821:virology; D011487:Protein Conformation; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral