Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1.
Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.
Dulude
Dominic
D
Département de Biochimie, Université de Montréal, 2900 Boulevard Edouard-Montpetit, Montréal, Québec H3T 1J4, Canada.
Baril
Martin
M
Brakier-Gingras
Léa
L
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017124:Conserved Sequence; D018965:Frameshifting, Ribosomal; D015685:Fusion Proteins, gag-pol / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics
D015685:Fusion Proteins, gag-pol; D012367:RNA, Viral; D008156:Luciferases