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 the viral nucleoprotein core (nucleocapsid) into a tightly coiled, helical nucleocapsid-M protein (NCM) complex. Using NCM complexes assembled in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein binding to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in their 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-sensitive M protein mutant lost the ability of M protein to reversibly dissociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast to the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaCl) 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 nucleocapsids rather than a change in soluble M protein. This result supports the idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.
Lyles
D S
DS
Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
McKenzie
M O
MO
D017022:Flow Injection Analysis; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D019251:Nucleocapsid / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D009994:Osmolar Concentration; D011485:Protein Binding / Q000187:drug effects; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000494:pharmacology; D014461:Ultracentrifugation; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000378:metabolism*
C094460:M protein, Vesicular stomatitis virus; D019590:Nucleocapsid Proteins; D014763:Viral Matrix Proteins; D012965:Sodium Chloride