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.

Extremely stable RNA structures can promote longterm ribosomal stalling or 'roadblocking', (35, 72) . In the dual-luciferase assay, roadblocking would result in decreased translation levels of the downstream firefly luciferase reporter gene product. However, the dual-luciferase assay controls for this, as frameshift efficiencies are normalized relative to in-frame control constructs (57, 67, 68) . Nevertheless, we asked if differential degrees of roadblocking might occur for our various constructs. The luminescence data reveal a consistent ratio of firefly/Renilla activity (data not shown) for all constructs (Table 1) . These values were calculated using the luminescence data from the positive control dual-luciferase constructs, where the Renilla and firefly genes are in frame and the slippery site is mutated such that the ribosome cannot frameshift. The consistency in the relative expression levels of the reporter genes indicates that roadblocking, if occurring, is uniform for all constructs.