Construction of chimeric virus clones and analysis of their sensitivity to neutralization by sCD4 and MAb b12. (A) The SalI/BamHI fragment of the LW_H8res gp120 envelope was inserted into the pLAI-2 vector, which contains the molecular clone of HIV-1 strain IIIB, to yield pLAI-H8. Site-directed mutagenesis was used to engineer three different variants of pLAI-H8 with amino acid substitutions at positions 164 and 370, pLAI-H8/E370, pLAI-H8/S164, and pLAI-H8/S164/E370 (the changes of A370 to Glu and N164 to Ser in the background of pLAI-H8 are depicted). In addition, the V1/V2 fragment of strain LW_G9res was placed into pLAI-H8, to yield pLAI-H8/G9V1V2, or into pLAI-H8/E370, to yield pLAI-H8/E370/G9V1V2. (B) Neutralization sensitivity of the chimeric molecular virus clones pLAI-H8 (open squares), pLAI-H8/G9V1V2 (open circles), pLAI-H8/E370/G9V1V2 (solid circles), pLAI-H8/E370 (solid squares), pLAI-H8/S164 (open triangles), and pLAI-H8/S164/E370 (solid triangles) for sCD4 and MAb b12. Virus stocks of the chimeric molecular clones were obtained by infection of PHA-stimulated PBMCs with supernatant from transfected 293T cells. Neutralization experiments were performed as described for Fig. 1.