Infection with live viruses was conducted in U373-MAGI-CXCR4 CEM and MT-2 cells. U373-MAGI-CXCR4 CEM cells were infected with normalized wild type or mutant live viruses in 24-well plate as described previously [21] . Briefly, triplicate wells (6610 4 cells/well) were infected with live viruses (60 pg p24/well) which were diluted in DMEM containing 20 mg/ml of DEAEdextran (Amersham Biosciences). After cultured at 37uC in 5% CO 2 incubator for 48 h, the cells were fixed in 1% formaldehyde-0.2% glutaraldehyde in PBS for 5 min. After washing twice with PBS, the cells were stained with 400 mg/ml of X-Gal (5-bromo-4chloro-3-indolyl-b-D-galactopyranoside), 4 mM MgCl 2 , 4 mM potassium ferrocyanide, and 4 mM potassium ferricyanide in PBS for 2 h at 37uC. The plate was washed twice with PBS and blue foci were observed under microscope. Infection with live viruses was also carried out in MT-2 cells as described previously [22, 23] . In brief, MT-2 cells (1610 4 cells/well) in 96-well plate were infected with live viruses (20 pg p24/well). After cultured for 6 days, the virus-specific cytopathic effect (CPE) was observed under microscope.