PARTIALLY FOLDED STATES OF THE CAPSID PROTEIN OF COWPEA SEVERE MOSAIC-VIRUS IN THE DISASSEMBLY PATHWAY

The different partially folded states of the capsid protein that appea r in the disassembly pathway of cowpea severe mosaic virus (CPSMV) wer e investigated by examining the effects of hydrostatic pressure, sub-z ero temperatures and urea. The conformational states of the coat prote in were analyzed by their intrinsic fluorescence, binding of bis(8-ani linonaphthalene-1-sulfonate) (bis-ANS) and susceptibility to trypsin d igestion. CPSMV could be disassembled by pressure at 2.5 kbar. Intrins ic fluorescence and hydrodynamic measurements showed that pressure-ind uced dissociation was completely reversible. Virus pressurization in t he presence of ribonuclease revealed that viral RNA was not exposed, s ince it was not digested by the enzyme, suggesting the maintenance of protein-nucleic acid interactions under pressure. When the temperature was decreased to -10 degrees C under pressure, CPSMV disassembly beca me an irreversible process and in this condition, viral RNA was comple tely digested by ribonuclease. These results suggest a relationship be tween protein-RNA interactions and CPSMV assembly. Bis-ANS binding and trypsin digestion of coat proteins revealed that they assume a differ ent conformation when they are denatured by low temperatures under pre ssure or than when they are denatured by urea at atmospheric pressure. The results indicate that the coat proteins can exist in at least fou r states: (1) The native conformation in the virus capsid; (2) bound t o RNA when the virus is dissociated by pressure at room temperature, a ssuming a conformation that retains the information for reassembly; (3 ) free subunits in a molten-globule conformation when the virus is dis sociated by low temperature under pressure; and (4) free subunits comp letely unfolded by high concentrations of urea. (C) 1997 Academic Pres s Limited.