Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system.
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.
Bacharach
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Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
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D001483:Base Sequence; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D019065:Virus Assembly
D015683:Gene Products, gag; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins