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.

The HIV-1 frameshift element variants containing the slippery sequence, intercodon and structural element of HIV-1 group M [35] were cloned into the pGL3s-hRLuc dual luciferase reporter vector [39] , containing a 5´human codon-optimised RLuc gene, the element, then a 3´Luc + gene in the −1 frame. A control element for normalisation of data contained a nullified slippery sequence (CUUCUGA) with the 3´Luc + gene in the original 0 frame such that output data reflect equimolar amounts of the two reporters synthesised. The human antizyme 1 (OAZ1) frameshift element of 120 nucleotides [7] was cloned into the dual luciferase vector but with the 3´Luc + in the +1 frame to ensure Luc + production only by a +1 frameshift. The same nullified construct (described above) was used for data normalisation. Constructs containing termination signals for readthrough assays were produced as described [39] , with the 3´Luc + gene in the original frame to ensure and measure continued translation only when termination failed. For data normalisation in this series, a construct containing the near-cognate UGG codon was used to produce equimolar amounts of both reporters. For the bicistronic fluorophore vector studies, the recoding sequences were excised and transferred into a vector between a 5´EGFP reporter and 3´DsRed.T4 reporter [40] in the appropriate frame [29] . eRF1 cDNA was cloned into the pcDNA3.1(+) vector (Invitrogen) using PCR primers (5´-ggaattcaagatggcggacgaccccagtgct-3´and 5´-gctctagactagtagtcatcaaggtcaaa-3´) to create pcDNA-eRF1. The suppressor tRNA vectors, ptRNAoc (UAA), ptRNAam (UAG) and ptRNAop (UGA), and the wild-type ptRNAser (serine) were made as described and were a kind gift from Dr Olivier Jean-Jean (Université Pierre et Marie Curie, Paris, France) [41] , [42] . Two siRNA target sequences [43] , si90 and si1186 (modified from si1187), were cloned into the shRNA expression vector pSilencer 3.0-H1 (Ambion) according to the manufacturer's instructions. A negative control (sh−ve), containing a sequence with limited homology to any portion of the human genome was provided by the manufacturer.