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. 2017 Oct 19;7(11):1815–1825. doi: 10.1002/2211-5463.12328

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© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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FRET assays for Gag–GagPol interactions with Gag interdomain constructs and virus particle production. (A) FRET efficiencies of Gag–GagPol with Gag interdomain fluorescent protein. HeLa cells were cotransfected with Gag(MA/EGFP/CA) plus GagPol(MA/mSB/CA), Gag(p2/EGFP/NC) plus GagPol(p2/mSB/NC), or Gag(p6/EGFP) plus GagPol(p6*/mSB/PR) molecular clone pair (a donor‐to‐acceptor DNA ratio of 1 : 1). Combinations of Gag(MA/EGFP/CA), Gag(p2/EGFP/NC), or Gag(p6/EGFP) molecular clone and a plasmid expressing soluble mSB were used as negative controls. The FRET efficiencies of the donor Gag–acceptor GagPol pairs were statistically greater than those of the corresponding negative controls (P < 0.01). *Statistically significant (P < 0.01) compared with the Gag(MA/EGFP/CA)–GagPol(MA/mSB/CA) pair. Representative FRET images are shown. (B) Virus particle production. HeLa cells were cotransfected with the Gag‐expressing and the GagPol‐expressing molecular clones at a DNA ratio of 10 : 1. The particle fractions purified from the cell culture supernatants were analyzed by western blotting using a monoclonal antibody specific for HIV‐1 p24CA.