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Journal of Virology, October 2000, p. 9515-9524, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein

Todd J. Green,1,2 Silvia Macpherson,1 Shihong Qiu,1,2 Jacob Lebowitz,1 Gail W. Wertz,1 and Ming Luo1,2,*

Department of Microbiology1 and Center for Biophysical Sciences and Engineering,2 University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 17 April 2000/Accepted 17 July 2000

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed 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 free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was 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 outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the 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 assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.

* Corresponding author. Present address: Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, 1918 University Blvd., MCLM 260, Birmingham, AL 35294. Phone: (205) 934-4259. Fax: (205) 934-0480. E-mail: ming{at}cmc.uab.edu.

Journal of Virology, October 2000, p. 9515-9524, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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