Figure 4

A premature termination codon inserted within the RSE at position 2685 underwent decay when readthrough of the gag stop codon was forced. A. Schematic of stop codon locations within the RSE. Premature termination codons were generated within the RSE at positions 2535, 2586, 2631, 2685 and 2736, named 1-5, respectively. The black font represents the minimal RSE as determined by truncations. Text in grey is RSE sequence flanking the minimal RSE. B. The RSE containing these mutations was cloned into two constructs, one with the natural gag termination codon sequence (WT gag stop) and the other with a single nucleotide insertion preceding the gag termination codon that forced readthrough into the pol open reading frame (Readthrough gag stop). Only PTC4 at position 2685 underwent decay in the readthrough construct. A representative RNase protection assay is shown, with the set of bands below each bar corresponding to the construct indicated directly above on the graph. Stars indicate a significant reduction compared to the wild-type virus (***: p < 0.0001)