Resolution of multiple complexes of phosphoprotein NS with nucleocapsid protein N of vesicular stomatitis virus.
The interaction of the nucleocapsid protein N and the phosphoprotein NS of vesicular stomatitis virus (VSV) was studied, free of other viral proteins, by transcription from SP6 vectors, followed by translation in a rabbit reticulocyte lysate. N-NS complex formation depended strongly on cotranslation of the two proteins; when N and NS were mixed following separate translation of each, very little complex formation occurred. Conditions were found under which at least six N-NS complexes were separated from each other by electrophoresis in a nondenaturing gel system, and the following findings were made. (i) These complexes fell into two groups; complexes 1 through 5 all had a stoichiometry of two molecules of N to one molecule of NS, whereas N-NS complex 6 had an equimolar ratio of the two proteins. (ii) N-NS complexes 1 through 5 predominated at lower concentrations of NS relative to N, but N-NS complex 6 was the major or sole product when NS was equimolar to or in excess of N. (iii) The two sets of complexes were formed by two distinct types of interactions of NS with N. The formation of N-NS complexes 1 through 5 was abolished by the removal of as few as 11 amino acid residues from the basic, highly conserved carboxy-terminal domain of NS, which is essential for the binding of NS to the N-RNA template of VSV. In contrast, formation of complex 6 was unaffected by removal of as many as 62 of the carboxy-terminal amino acids of NS, a region encompassing both the terminal basic domain and an adjacent domain which is required for VSV RNA polymerase function. The significance of these observations for the mechanism of VSV genome replication is discussed.
Masters
P S
PS
Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Banerjee
A K
AK
D002213:Capsid / Q000502:physiology*; D046911:Macromolecular Substances; D010750:Phosphoproteins / Q000502:physiology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology*; D014758:Viral Core Proteins / Q000502:physiology*; D017361:Viral Nonstructural Proteins
D046911:Macromolecular Substances; D010750:Phosphoproteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins