Figure 2

Quantification of 1-LTRc. (A) Possible amplifications from the various substrates found in infected cells with primers used for 1-LTRc quantification. 1: 1-LTRc; 2: 2-LTRc and 3: linear viral DNA. (B) Plasmids (1: p1-LTR and 2: p2-LTR) used as controls. Linear DNA (3) was obtained by ScaI digestion of p2-LTR. (C) Amplification of serial dilutions of p1-LTR using the protocol for 1-LTRc amplification. Amplification of known amounts of p1-LTR (1), p2-LTR (2) and linear DNA (3) using the protocol for 1-LTRc quantification. The results are reported in the table, for an elongation time of 25 s. Input: initial amount of target. Output: amount measured with the 1-LTRc protocol. %Amplification calculation is based on the output:input ratio. The plot shows the amplification results with known amounts of p1-LTR. The PCR products obtained with the various substrates were loaded onto an agarose gel (shown below the table). M: Molecular weight marker. (D) 1-LTRc quantification is not influenced by the presence of 2-LTRc. Nalm6 and Nalm114 cells were infected with VSV-G-pseudotyped NLENG1-ES-IRES WT (left panel) or NLENG1-ES-IRES D116N (right panel). At different times post-infection, the percentages of 2-LTRc and 1-LTRc were determined in Nalm6 (ligase-4+) (black columns) and Nalm114 (ligase-4-) (white columns) cell lines. Each value corresponds to an average of five to six independent experiments and confidence intervals analysis are shown for a p value <0.05.