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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 interactions of Gag and GagPol with mutations and virus particle production. (A) FRET efficiencies of Gag(MA/EGFP/CA) and GagPol(MA/mSB/CA) containing mutations. HeLa cells were cotransfected with Gag(MA/EGFP/CA) plus GagPol(MA/mSB/CA) molecular clone containing CA mutations (W184A and M185A) or a deletion of the CA CTD (ΔCA CTD), NC (ΔNC), or SL1 and 3 (ΔSL1,3) at a DNA ratio of 1 : 1. A combination of Gag(MA/EGFP/CA) and soluble mSB was used as the negative control. WM > AA, W184A and M185A. The FRET efficiencies of the donor Gag–acceptor GagPol pairs were statistically greater than that of the negative control (P < 0.01). *Statistically significant (P < 0.01) compared with the wild‐type Gag(MA/EGFP/CA)–GagPol(MA/mSB/CA) pair. Representative FRET images are shown. (B) Virus particle production. HeLa cells were cotransfected with Gag(MA/EGFP/CA) and GagPol(MA/mSB/CA) 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.