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.

We have used a reporter containing two luciferase-coding open reading frames, the second oriented in the −1 frame with respect to the first, and with the HIV-1 frameshift element placed between them, to study the function of the element in cultured cells. Specifically, the role of GGG, found immediately after the slippery sequence, has been evaluated for its role in frameshifting in HIV-1. This 'intercodon' is almost as highly conserved as the slippery sequence itself and is more conserved than the other nucleotides of the stem-loop (Fig. 1) . The primary role of this intercodon in supporting −1 PRF efficiency does not appear to be either maintaining the structure of the lower stem-loop, or stacking of its first nucleotide on the last codon of the slippery sequence ( Fig. 2 and Fig. 3) . A strategy to test whether this intercodon has as its primary function an involvement in decoding before frameshifting utilised a stop codon substitution of the GGG intercodon. Both protein and tRNA decoding molecules for stop signals were then used to probe the decoding function of the intercodon. In this system, over-expression of the cognate decoding protein, eRF1, caused a reduction in-1 PRF (Fig. 5 ). This implied that the intercodon occupied the ribosomal A site and was available for decoding prior to at least a proportion of frameshift events. Suppressor tRNAs mitigated eRF1-induced reduction in −1 PRF efficiency, reinforcing the conclusion that the observed effects of eRF1 were specific to decoding activity in the A site of the ribosome.