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.

DNA templates used for the dual-luciferase frameshift assay were cloned into a p2luc vector between the rluc and fluc reporter genes. Briefly, complementary synthetic oligonucleotides [Integrated DNA Technologies (IDT), Inc.] with BamH I and Sac I compatible ends were cloned into the p2luc vector using the BamH I and Sac I sites between the rluc and fluc reporter genes. Oligonucleotides comprising the template sequences (Supplementary Table S1 ) and their complements were phosphorylated, annealed and ligated into the p2luc vector to produce the experimental constructs. This places the fluc gene in the À1 reading frame relative to rluc; analogous to the orientation of the gag and pol genes in the HIV-1 genome. For the spacer mutation constructs (MS13-17), a compensatory number of nts were added or removed downstream of the frameshift site to maintain the appropriate reading frame of the downstream reporter gene. The 'wild-type' (WT) sequence utilized here corresponds to the most frequently occurring sequence found in HIV-1 group M subtype B NL4-3 laboratory strain (56) . Positive control sequences and their complements were also cloned into the p2luc vector and have two thymidine residues (Supplementary Table S1 , bold) in the slippery sequence (Supplementary Table S1 , underlined) replaced with cytidines, and an additional nt inserted immediately before the Sac I complementary sequence (GAGCT), which places the rluc and fluc genes in-frame. In all constructs, a Pml I restriction site was included at the end of the template to allow for run-off transcription after digestion with the Pml I enzyme (NEB). Resultant products were transformed into Escherichia coli competent cells (DH5a). Plasmid DNA was purified from cell cultures (Qiagen) and the sequences of all constructs were verified (University of Wisconsin-Madison Biotechnology Center).