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J Virol, August 1998, p. 6944-6949, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Binding of the Human Immunodeficiency Virus Type 1 Gag Protein to the Viral RNA Encapsidation Signal in the Yeast Three-Hybrid System

Eran Bacharach and Stephen P. Goff*

Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032

Received 29 January 1998/Accepted 12 May 1998

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi ). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.

* Corresponding author. Mailing address: Room 1127 Hammer Health Science Center, Columbia University P & S, 701 West 168th St., New York, NY 10032. Phone: (212) 305-7965. Fax: (212) 305-8692. E-mail: goff{at}cuccfa.columbia.edu.

J Virol, August 1998, p. 6944-6949, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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