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.

During HIV-1 evolution the GGG intercodon may have been selected to give a level of frameshifting that produces a ratio of enzyme to structural proteins that best supports virus replication. The work presented in this study supports the hypothesis that −1 PRF in HIV-1 occurs relatively frequently with the slippery sequence at the E and P sites, and highlights that events during decoding of the intercodon are critical for frameshift efficiency. Namely, competition between decoding the GGG intercodon (resulting in regular translation with a maintained reading frame) and −1 PRF occurs in a portion of HIV-1 frameshift events. This scenario can be integrated into existing models of frameshifting and supports the notion that frameshifting in HIV-1 is a heterogeneous process. [29] . 'Null0' refers to an in-frame control reporter, and 'controls' refers to background fluorescence values from mock-transfected cell lysate. The mean ± SEM frameshift efficiencies for three replicates from one experiment are shown in the included graph. (XLSX) S2 File. Raw data relating to Fig. 4 and alternative shRNA. A. Quantitative PCR. Threshold cycle (Ct) values for target gene (TG) eRF1 and reference gene (RG) 18S rRNA. Two pooled replicates were tested, each containing cell lysate from four individually transfected wells (a total of eight transfections). Three technical replicates were made for each pooled sample. Data were analysed using the comparative CT method to give fold differences and standard deviations. B, baseline; T, threshold; NTC, no template control; 0ug, non-transfected control. '-ve', '90' and '1186' correspond to the shRNA vectors that were transfected. C. Stop codon readthrough. Values for Renilla luciferase (Rluc) and firefly luciferase (Luc+) are in relative light units. Background (bkgd) values (cell lysate alone) were subtracted from each measurement. The percentage of readthrough of the stop signal for each sample was calculated using the corresponding sense readthrough control (RT) from each replicate plate. Replicates are from three individual experiments. '-ve', '90' and '1186' correspond to the shRNA vectors that were transfected. D. Antizyme frameshifting. Values for Renilla luciferase (Rluc) and firefly luciferase (Luc+) are in relative light units. Background (bkgd) values (cell lysate alone) were subtracted from each measurement. The relative frameshift efficiency for each sample was calculated using the corresponding average readthrough control from each replicate plate. Replicates are from one experiment. The value in bold italics was not included as it was greater than two standard deviations above the mean. Az, antizyme; RT, readthrough control. '-ve', '90' and '1186' correspond to the shRNA vectors that were transfected. E. HIV frameshifting. Values for Renilla luciferase (Rluc) and firefly luciferase (Luc+) are in relative light units. Background values for target gene (TG) eRF1 and reference gene (RG) 18S rRNA. Two pooled replicates were tested, each containing cell lysate from four individually transfected wells (a total of eight transfections). Three technical replicates were made for each pooled sample. Data were analysed using the comparative CT method to give fold differences and standard deviations. B, baseline; T, threshold; NTC, no template control. C. Stop codon readthrough. Values for Renilla luciferase (Rluc) and firefly luciferase (Luc+) are in relative light units. Background (bkgd) values (cell lysate alone)