The parental HIV-1 proviral plasmids, pLAI.2, pNL4-3-DE-EGFP and pHEF-VSV-G, were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [18] [19] [20] . The pNL4-3-DE-EGFP based mutants (Y271A, G273A, I274A, K275A, V276A, R277A) and pLAI2 based mutants (Y271A and I274A) were constructed by sitedirected mutagenesis using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, USA) according to manufacturer's instruction. The open reading frame of RT p66 subunit was amplified from wild type or mutant pNL4-3-DE-EGFP using a pair of primers and cloned into pET-28b vector (Novagen, Shanghai) to obtain expression plasmid pET-p66 with the 66 His tag at 39 terminus. The paired primers used for site-directed mutagenesis and construction of plasmids are listed in Table 1 . Cell lines 293FT, HeLa and U373-MAGI-CXCR4 CEM [21] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics (Invitrogen, USA). MT2 cells [22, 23] were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics.