REVERSIBLE AND IRREVERSIBLE STEPS IN ASSEMBLY AND DISASSEMBLY OF VESICULAR STOMATITIS-VIRUS - EQUILIBRIA AND KINETICS OF DISSOCIATION OF NUCLEOCAPSID-M PROTEIN COMPLEXES ASSEMBLED IN-VIVO

The matrix (M) protein of vesicular stomatitis virus (VSV) condenses t he viral nucleoprotein core (nucleocapsid) into a tightly coiled, heli cal nucleocapsid-M protein (NCM) complex. Using NCM complexes assemble d in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein bindi ng to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in t heir M proteins were analyzed using sedimentation and light-scattering assays. At physiological ionic strength, the binding reaction had the characteristics of a dynamic reversible equilibrium. A temperature-se nsitive M protein mutant lost the ability of M protein to reversibly d issociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast t o the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaC l) were not able to bind M protein at low ionic strength with the same high affinity seen in NCM complexes assembled in vivo. The effect of incubation at 250 mM NaCl was shown to be due to a change in nucleocap sids rather than a change in soluble M protein. This result supports t he idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.