Rgk. Donald et Ao. Jackson, RNA-BINDING ACTIVITIES OF BARLEY STRIPE MOSAIC-VIRUS GAMMA-B FUSION PROTEINS, Journal of General Virology, 77, 1996, pp. 879-888
The barley stripe mosaic virus (BSMV) gamma b gene encodes a 17 kDa cy
steine-rich protein known to affect virulence and to have a role in re
gulating viral gene expression. We have constructed recombinant gamma
b-glutathione S-transferase fusion proteins in Escherichia coli and ha
ve determined the ability of the purified fusion proteins and various
mutant derivatives to bind nucleic acids in vitro. Gel-shift analyses
revealed that the wild-type gamma b-fusion protein is able to bind RNA
cooperatively. The binding affinity is highly selective for single-st
randed RNA because double-stranded RNA, single-stranded and double-str
anded DNA, and transfer RNA were unable to compete for binding with th
e labelled RNA probes. However, BSMV-specific sequence binding was not
observed since a chloroplast RNA competed for binding with P-32-label
led transcripts derived from the BSMV genome. The first 44 amino acids
of the 152 amino acid gamma b fusion protein encompassing one of two
cysteine-rich 'zinc finger-like' moths, and a basic region separating
the finger-like moths are required for RNA binding. Site-specific amin
o acid substitutions within two groups of lysine and arginine residues
located in the basic moth reduced the binding affinity of the fusion
protein greatly, but cysteine and histidine substitutions designed to
disrupt the finger-like moths failed to have appreciable effects on bi
nding. These findings indicate that the regulatory properties of gamma
b may be mediated in part by RNA binding activities.