In vitro analysis of an RNA binding site within the N-terminal 30 amino acids of the southern cowpea mosaic virus coat protein.

Southern cowpea mosaic virus (SCPMV) is a positive-sense RNA virus with T = 3 icosahedral symmetry. The coat protein (CP) has two domains, the random (R) domain and the shell (S) domain. The R domain is formed by the N-terminal 64 amino acids (aa) and is localized to the interior of the particle where it is expected to interact with the viral RNA. The R domain (aa 1--57) was expressed in Escherichia coli as a recombinant protein (rWTR) containing a nonviral C-terminal extension with two histidine tags. The RNA binding site of the R domain was identified by Northwestern blotting and electrophoretic mobility shift assay (EMSA) using recombinant wild-type and mutant R domain proteins. Deletions within the R domain revealed that the RNA binding site is localized to its N-terminal 30 aa. RNA binding by this element was found to be nonspecific with regard to RNA sequence and was sensitive to high salt concentrations, suggesting that electrostatic interactions are important for RNA binding by the R domain. The RNA binding site includes 11 basic residues, eight of which are located in the arginine-rich region between aa 22 and 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21, did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an alpha-helix in the presence of 50% trifluoroethanol (TFE). The possible role for this structural change in RNA binding by the R domain is discussed.