Construction of NL4-3 mutants differing in coreceptor usage. A, Amino acid V3 loop sequence of the NL4-3 laboratory strain and of two primary isolates PI-952 (R5X4-dualtropic) and PI-991 (R5-monotropic) of different phenotype. B, V3 loop regions of patient isolates were generated by PCR amplification followed by a second PCR. In the second PCR amplification silent mutations were introduced to generate the BglII and NheI restriction sites. PCR fragments were cloned into pUCenv-deltaV3 and the NL4-3 env – V3 loop chimera was cloned into the pNL4-3Bst retroviral vector as a BstEII/BamHI fragment. C, This V3 cloning procedure was carried out to generate mutants of NL4-3, designated NL-952 and NL-991, containing the V3 loop and the coreceptor phenotype of the of the original primary isolates. NL4-3, X4-monotropic; NL-952, R5X4-dualtropic; NL-991, R5-monotropic.