Yeast Two-Hybrid Screening of HIV-1 Gag

The infectious molecular clone pNL-CH [33 (link)], which was derived from the pNL₄₋₃ clone of HIV-1, was a gift from R. Swanstrom. To perform Y2H screening, the Pr55^gag and CA coding region of the HIV-1_NL–CH were cloned into pGBKT7 (bait) (Clontech Laboratories, Inc.) as previously described [34 (link)].To prepare the ATE1-V5 expression vector, the coding region of ATE1, which was amplified by PCR using the primers ATE1-TCF (5′-AAAGCTTGCCATGGCTTTCTGGGCGGGG-3′) and ATE1-TCR (5′-TCTCGAGCAGTTTCTGAACAGCAGCATCCG-3′), was cloned into the pcDNA™ 3.1D/V5-His-TOPO^® vector (Thermo Fisher Scientific, Inc.).