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
Ds. Lyles et Mo. Mckenzie, 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, Biochemistry, 37(2), 1998, pp. 439-450
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.