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. 1998 Aug;72(8):6944-9.
doi: 10.1128/JVI.72.8.6944-6949.1998.

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system

E Bacharach  1 , S P Goff
Affiliations

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system

E Bacharach et al. J Virol. 1998 Aug.

Abstract

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.

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Figures

FIG. 1
FIG. 1
(A) General scheme of the three-hybrid system used in this study, adapted from reference . Binding of the RNA hybrid to the LexA-MS2 coat protein and to the Gal4AD-HIV Gag protein leads to formation of a complex which activates transcription of the lacZ gene. The yeast strain, L40-coat, used in this study constitutively expresses the LexA-MS2 coat fusion protein from an integrated gene (46). (B) Structure of the RNA hybrid. Shown from 5′ to 3′ are the stem-loop RPR1 leader (84 nt), linker (30 nt), the four stem-loops of the HIVΨ (139 nt), linker (23 nt), two stem-loop structures that bind the MS2-coat protein (60 nt), and the RPR1 3′ terminal sequence (41 nt). The structures have not been drawn to scale. (C) HIVΨ RNA sequence, adapted from reference . The alternative positions of the cytosine insertion are marked with arrows. The location of the HIV-1 splicing donor (SD) is marked with an arrowhead, and the translation start codon appears in bold letters. Each of the stem-loops is marked by a number (SL1 to SL4).
See this image and copyright information in PMC

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