Study of the assembly of vesicular stomatitis virus N protein: Role of theP protein

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P prot ein) were expressed together in Escherichia coli, The N and P proteins form ed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA, A soluble N protein-RNA oligomer fr ee of the P protein was isolated from the N/P protein-RNA complex using con ditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it wa s composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with o uter diameter, inner diameter, and thickness of 148 +/- 10 Angstrom, 78 +/- 9 Angstrom, and 83 +/- 8 Angstrom, respectively, as determined by electron microscopy, RNA in the complexes was protected from RNase digestion and wa s stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation, In addition to coexpression with th e full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for a ssembly of N protein-RNA oligomers. With the results provided in this repor t, we propose a model for the assembly of an N/P protein-RNA oligomer.