Fig. 4

Functional activity of selected RNA elements in a transient transfection assay. The functional activities of eleven of the RcREs identified with the targeted bioinformatic approach were measured by transfecting the reporter constructs containing each RcRE into 293T/17 cells together with either 50 ng of prRev or prRec or no additional vector. As controls, reporter constructs containing the prRcRE and HIV-1 RRE were also tested. In addition, a plasmid expressing Secreted Alkaline Phosphatase (SEAP) was transfected with each sample as a normalization control for transfection efficiency. After 72 h, cell supernatants were harvested and supernatant p24 and SEAP were measured. The samples were then normalized for differences in transfection efficiency using the SEAP values. For comparison between experiments the SEAP normalized p24 value for the Rec/prRcRE pair was set to 1 and the activity of each Rev/RcRE or Rec/RcRE combination relative to this normalized value was plotted. The error bars show the variation between two independent transfections performed at the same time. To determine p values, between Rev and Rec, a two-tailed student t-test was performed using GraphPad Prism 8. The results shown in a, b were from transfections performed on different days. In a, the raw prRec/prRcRE p24 value before normalization was 6.4 ng/ml and in b it was 4.8 ng/ml