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Journal of Virology, January 1999, p. 19-28, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

David E. Ott,* Elena N. Chertova, Laura K. Busch, Lori V. Coren, Tracy D. Gagliardi, and Donald G. Johnson

AIDS Vaccine Program, SAIC/Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

Received 2 July 1998/Accepted 11 September 1998

The p6Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6Gag between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6Gag proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

* Corresponding author. Mailing address: AIDS Vaccine Program, SAIC/Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Building 535, Room 433, Box B, Frederick, MD 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}avpvx1.ncifcrf.gov.

Journal of Virology, January 1999, p. 19-28, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.