title: Structure-function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors.
abstract: Expression of the pol-encoded proteins of human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift at the junction of the gag and pol coding sequences. Frameshifting takes place at a heptanucleotide slippery sequence, UUUUUUA, and is enhanced by a stimulatory RNA structure located immediately downstream. In patients undergoing viral protease (PR) inhibitor therapy, a p1/p6(gag) L449F cleavage site (CS) mutation is often observed in resistant isolates and frequently generates, at the nucleotide sequence level, a homopolymeric and potentially slippery sequence (UUUUCUU to UUUUUUU). The mutation is located within the stimulatory RNA downstream of the authentic slippery sequence and could act to augment levels of pol-encoded enzymes to counteract the PR deficit. Here, RNA secondary structure probing was employed to investigate the structure of a CS-containing frameshift signal, and the effect of this mutation on ribosomal frameshift efficiency in vitro and in tissue culture cells was determined. A second mutation, a GGG insertion in the loop of the stimulatory RNA that could conceivably lead to resistance by enhancing the activity of the structure, was also tested. It was found, however, that the CS and GGG mutations had only a very modest effect on the structure and activity of the HIV-1 frameshift signal. Thus the increased resistance to viral protease inhibitors seen with HIV-1 isolates containing mutations in the frameshifting signal is unlikely to be accounted for solely by enhancement of frameshift efficiency.
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Roseanne
authors.initials: R
authors.lastname: Girnary
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: King
authors.affiliation: GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
authors.forename: Laurence
authors.initials: L
authors.lastname: Robinson
authors.affiliation: GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Elston
authors.affiliation: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Brierley
mesh_terms: D024882:Drug Resistance, Viral / Q000235:genetics*; D018965:Frameshifting, Ribosomal; D015967:Gene Expression Regulation, Viral; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics
chemical_list: D017320:HIV Protease Inhibitors; D012367:RNA, Viral