The Highly Conserved Codon following the Slippery Sequence Supports -1 Frameshift Efficiency at the HIV-1 Frameshift Site

Mathew, Suneeth F.; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S.; McKinney, Cushla; Poole, Elizabeth S.; Tate, Warren P.

Surprisingly, although no change was expected, the frameshift efficiency increased substantially with the naturally occurring HIV-1 PRF element (GGG intercodon) in response to shRNA expression independent of shRNA sequence. Frameshift efficiency increased 2-3 fold to 20-25% with both α-eRF1 and the shRNA negative control sequences (Fig. 4E) . This was consistent with a previously observed general increase in −1 PRF upon dosage with small RNAs [55] . Non-physiological artefacts caused by shRNA expression could mask any specific The effect of eRF1 depletion on termination and recoding. A. eRF1 transcript levels measured using quantitative real-time PCR. Results show eRF1 transcripts within RNA isolated from non-transfected HEK293T cells ('none'), and eRF1 transcripts within RNA isolated from those transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The mean ± standard deviation (SD) for six replicates is shown. B. Immunoblot of eRF1 in protein extracts from non-transfected HEK293T cells ('none'), or cells transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The ratios were calculated after normalisation to β-actin in each case and compared with the non-transfected control (1.0). Raw data is available in S1 Fig. C . Readthrough at a UGA test context (UGACAG). A dual luciferase construct with the two reporters in the same frame and the test stop signal separating them was co-transfected with the control and α-eRF1 shRNAs. Readthrough at the test stop signal was determined in each case (α-eRF1 and − control.) The mean ± SEM for 12 replicates from three individual experiments is shown. D. Effect of depletion of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for eight replicates is shown. E. Effect of eRF1 depletion on frameshifting with the native GGG intercodon and substituted stop codon (UGA or UAG). The mean ± SEM for a minimum of 10 replicates from at least two independent experiments is shown. A dotted line indicates the level of −1 PRF in the absence of any shRNA (see Fig. 2 ). **P = < 0.01, ***P = < 0.001, n.s., not significant. effects of depleting eRF1 for the −1 PRF test system, in contrast to decoding competition at stop signals (readthrough and +1 PRF). Alternatively, a *2-fold reduction in the eRF1 protein observed may be insufficient to perturb the balance of forces promoting or opposing frameshifting. Indeed, the UGA and UAG intercodon constructs are not apparently affected by the shRNAs, with a typical frameshift efficiency of 4-5% recorded (Fig. 4E) .