The RT activity in pseudoviruses was measured by a RT assay using colorimetric kit (Roche, USA). Briefly, viral supernatants containing 2 mg p24 were centrifuged at 4uC for 2 h at 40,0006g and the viral pellets were resuspended in 50 ml lysis buffer. Lyzed viral pellets were 10 fold serially diluted and the subsequent procedures were carried out according to the manufacturer's instructions. RT activities of mutants were calculated and compared to that of wild type pseudovirus.