Journal of Virology, November 2000, p. 10260-10268, Vol. 74, No. 21
Department of Molecular Biology and Genetics,
Cornell University, Ithaca, New York 14853
Received 2 May 2000/Accepted 27 July 2000
Purified retrovirus Gag proteins can assemble in vitro into
virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the
protease domain forms spherical particles resembling immature virions
lacking a membrane but that a similar protein missing the p10 domain
forms tubular particles. Thus, p10 plays a role in spherical particle
formation. To further study this shape-determining function, we
dissected the p10 domain by mutagenesis and examined VLPs assembled
within Escherichia coli or assembled in vitro from purified
proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to
restore efficient spherical assembly to a p10 deletion mutant. Random
and site-directed mutations were introduced into this segment of
polypeptide, and the shapes of particles formed in E. coli
were examined in crude extracts by electron microscopy. Three
phenotypes were observed: tubular morphology, spherical morphology, or
no regular structure. While the particle morphology visualized in crude
extracts generally was the same as that visualized for purified
proteins, some tubular mutants scored as spherical when tested as
purified proteins, suggesting that a cellular factor may also play a
role in shape determination. We also examined the assembly properties
of smaller Gag proteins consisting of the capsid protein-nucleocapsid
protein (CA-NC) domains with short N-terminal extensions or deletions.
Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low.
Deletion of the N-terminal residue(s) abrogated assembly. Taken
together, these results imply that the N terminus of CA and the
adjacent upstream 25 residues play an important role in the
polymerization of the Gag protein.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of the Rous Sarcoma Virus p10 Domain in Shape
Determination of Gag Virus-Like Particles Assembled In Vitro and
within Escherichia coli
and
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, Biotechnology Bldg., Cornell
University, Ithaca, NY 14853. Phone: (607) 255-2443. Fax: (607)
255-2428. E-mail: vmv1{at}cornell.edu.
Present address: Department of Medicine (Infectious Diseases),
University of Massachusetts Medical School, Worcester, MA 01655.
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