Activation of cryptic splice sites by synonymous mutations in Gag.

<p>(A, B) MT4 cells were infected with the indicated virus (harvested from the supernatant of 293T cells transfected proviral plasmids representing each of the WT(HIV-1_NHG), mutant (A, B and revertants (A C819T, A1130G and B T1311C, G1326A) thereof) at an MOI of 0.002. Aliquots of infected cells were withdrawn each day and the proportion of infected cells determined by FACS analysis of GFP expression. (C) Next gen sequencing analysis of HIV-1 splicing. The heatmap indicates relative proportion of sequencing reads that used the cryptic splice sites for WT(HIV-1_NHG), mutant (A, B and revertants thereof). (D,E) Schematic representation of the mutant blocks of nucleotides in HIV-1 mutants A (D) and B (F), indicating positions of mutant derivatives (AA, AB, BA, BB) etc, and the positions of cryptic splice sites and revertant mutant sites. Blocks colored blue are those that conferred overt splicing perturbations when mutated. (F) Fluorescent primer PCR analysis of HIV-1 splicing in mutant A. A sense PCR primer situated 5’ to the cryptic donor D1169, was used along with an antisense primer positioned 3’ to A7 (labelled with IRD800) was used to amplify cDNAs derived from the 1.8 kb class of spliced HIV-1 mRNAs respectively. (G) Fluorescent primer PCR analysis of HIV-1 splicing in mutant B. A sense PCR primer situated 5’ to D1, was used along with an antisense primer positioned 3’ to the mutant B block (labelled with IRD800) was used to amplify cDNAs derived HIV-1 mRNAs. For panels (F) and (G) PCR products were subjected to PAGE and a LI-COR Odyssey scanner was used to detect fluorescent signals directly from the gels.