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Background:  The synthesis of the HIV-1 Gag-Pol polyprotein, precursor of the viral enzymes, results from a programmed -1 ribosomal frameshift event when ribosomes translate the full-length viral messenger RNA. Translation of the same messenger, according to conventional translation rules, generates Gag, the precursor of the structural proteins of the virus. The frameshift event occurs with a defined efficiency that appears to be critical for the production of infectious particles and is controlled by an RNA structure motif following the slippery sequence where the frameshift occurs. We hypothesize that mutations in this motif affect frameshift efficiency, and that this perturbation of the Gag/Gag-Pol ratio influences the virus infectivity and replication rate.

Methods:  Our experimental approach consists of introducing mutations within the frameshift signal that increase or decrease the efficiency of frameshift and in investigating the effects of these mutations on viral assembly and replication. The effects on viral production and release are assessed by measuring the capacity of COS7 cells transfected with a plasmid containing the full-length gag-pol gene of HIV-1 under control of an SV40 promoter to produce HIV virus-like particles. Quantification of Gag (with a p24 ELISA) and Gag-Pol (with a reverse transcriptase enzymatic assay) inside the cells and in the supernatant can discriminate between release and assembly defects, respectively, whereas the infectivity of the frameshift mutants is assessed in single-round infection assays using an indicator cell line that carries the β-galactosidase gene under the control of the HIV promoter LTR.

Results:  Our studies with the virus-like particles produced with the Gag-Pol expressor vector show that a decrease or an increase of 50% in the frameshift efficiency did not alter the release of virus-like particles, but reduced the level of Gag-Pol (reverse transcriptase) incorporated in these virus-like particles, suggesting a defect in maturation/infectivity, which was confirmed by single-round infection assays with proviral constructs.

Conclusions:  The important consequences of minor variations in the frameshift efficiency stress the potential of the frameshift event as a novel target for anti-HIV drugs.

Keywords: frameshift signal; potential therapeutic target; mutational analysis