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Journal of Virology, October 2002, p. 10226-10233, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10226-10233.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

Steve C. Pettit,1,{dagger} Gavin J. Henderson,1,2 Celia A. Schiffer,3 and Ronald Swanstrom1*

UNC Center for AIDS Research,1 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295,2 Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester, Massachusetts 016553

Received 7 January 2002/ Accepted 28 June 2002

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1' amino acid. These results point to a trans effect between the P1 and P1' amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

* Corresponding author. Mailing address: CB7295, Rm. 22-006 Lineberger Bldg., UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: (919) 966-5710. Fax: (919) 966-8212. E-mail: risunc{at}med.unc.edu.

{dagger} Present address: CB7030, Department of Medicine, Infectious Disease Division, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7030.

Journal of Virology, October 2002, p. 10226-10233, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10226-10233.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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