1. ERT and uncoating assay shows increased vector capsid uncoating upon successful central DNA Flap formation.
ERT and uncoating assay shows increased vector capsid uncoating upon successful central DNA Flap formation. (A) ERT was carried out on vector supernatants, and qPCR was used to quantify late RT° products and early minus strong stop DNA ((−)ssDNA) products (upper panel). The qPCR quantification was carried out in triplicate and is highly representative of nine independent experiments carried out on three distinct vector productions. ERT reactions were then layered onto sucrose cushion to isolate monomeric CA proteins, indicative of productive uncoating, which were quantified by p24 ELISA assay (lower panel). The p24 ELISA quantification is represented as a percentage of monomeric CA obtained for the corresponding Triton X‐100‐treated vector samples (% uncoating), and is a mean of three independent experiments carried out on three distinct vector productions, representative of five independent experiments. (B) Southern blotting using a vector specific probe was used to monitor the presence of full‐length reverse‐transcribed products in the ERT reactions compared with Hirt samples. The radiograph shown is representative of three independent experiments. (C) Formation of the central DNA Flap in Flap+ samples was assessed by dC‐tailing of the 3′ (+) strand overlap followed by qPCR using primers specific for the ter2‐dCtail region and a 5′ region ∼200 bp upstream (upper panel). The number of copies of dC‐tailed amplified products is represented as a percentage of late RT° products (lower panel). The quantification was carried out in triplicate and is highly representative of five independent experiments.
Nathalie J Arhel et al. EMBO J. 2007;26:
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