RNA-protein interactions and secondary structures of cowpea chlorotic mottle virus for in vitro assembly.
Laser Raman spectroscopy of the cowpea chlorotic mottle virus (CCMV) in native (pH 5.0) and partially swollen (pH 7.5) states reveals the presence of small percentages of protonated adenine (less than 15%) and cytosine (less than 7%) bases in the encapsidated RNA molecule of the native virion. The protonated bases are titrated with pH-induced swelling of the virus. Titration of putative COOH groups of aspartic and glutamic side chains of the virion subunit cannot be detected over the same pH range, which suggests that carboxyl anions (CO-2) and protonated bases are both available at pH 5 to stabilize the ribonucleoprotein particles by electrostatic interactions. The highly (95%) ordered secondary structure of encapsidated RNA may undergo a small additional increase (less than 3%) in ordered structure with release from the virion, suggesting at most a marginal structure-distorting influence from protein contacts in the native particle. The Raman spectra of the virion are also compared by difference spectroscopy with spectra of capsids (empty shells devoid of RNA), subunit dimers, and protein-free RNA. The results indicate that the subunit structure is altered by the release of RNA from the virion, as well as by the swelling of the virion. Amino acid residues and protein secondary structures that are affected in these in vitro assembly and disassembly processes are identified from their characteristic Raman lines. Two classes of cysteinyl SH groups, solvent exposed and solvent protected, are revealed for the capsid and virion subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D002213:Capsid / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D012328:RNA Viruses / Q000648:ultrastructure*; D012367:RNA, Viral / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D014764:Viral Proteins / Q000378:metabolism*; D014771:Virion / Q000648:ultrastructure
D000596:Amino Acids; D046911:Macromolecular Substances; D012367:RNA, Viral; D014764:Viral Proteins