HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome

Mouzakis, Kathryn D.; Lang, Andrew L.; Vander Meulen, Kirk A.; Easterday, Preston D.; Butcher, Samuel E.

Within viral capsids, the HIV-1 frameshift site RNA is part of a conserved 3HJ secondary structure ( Figure 6A ) (54, 55) . It has been hypothesized that the role of this secondary structure is to slow down the rate of translation (54) , which in turn may modulate frameshift efficiency. We therefore compared the 3HJ secondary structure (3HJ WT) to a similar construct with mutations designed to disrupt secondary structure formation in the P1 and P2 helices ( Figure 6B , 3HJ Mut). We observe a significant decrease in frameshift efficiency, from 4.6 ± 0.5% to 2.5 ± 0.4%, when the 3HJ secondary structure is present ( Figure 6C and Table 1 , compare 3HJ WT to 3HJ Mut). As expected, there is no significant difference between the observed frameshifting efficiencies of the 3HJ Mut and the WT construct used above (4.7 ± 0.5 and 4.6 ± 0.5, respectively). The observed frameshift efficiencies for our 3HJ WT and WT reporter constructs in RRL both fall within the range of previously measured frameshifting efficiencies for HIV-1 in vivo, which range from 2% to 5% (9, 15, 21, 24 ). Next, we tested the local stability hypothesis in the context of the 3HJ secondary structure by increasing the local stability of 2 bp in the P3 helix ( Figure 6A ). The 2-bp change (3HJ MS1) results in a large, 5-fold increase in frameshift efficiency ( Figure 6C and Table 1 ). This increase is similar to the 4-fold difference between the MS1 and WT RNAs ( Figure 3A and Table 1 ). We conclude that the 3HJ secondary structure indirectly modulates frameshifting, likely by altering the kinetics of translation, as previously hypothesized (54) . This effect must happen prior to frameshifting, as the ribosome disrupts the 3HJ secondary structure as it encounters the slippery site. Once the ribosome is engaged with the slippery site, local stability is the primary determinant of frameshifting efficiency, as illustrated by comparison of 3HJ WT to 3HJ MS1 ( Figure 5C ).