Figure 2
Long-term culture of W596L/K601D virus. (A) Wild type and W596L/K601D-mutated HIV-1AD8 virus stocks produced by transfected 293T cells were normalized according to RT activity and used to infect U87.CD4.CCR5 cells. The cell-free virus obtained at day 10 was filtered through a 0.45 μm nitrocellulose filter, normalized according to RT activity and used to infect fresh U87.CD4.CCR5 cells. Viruses were subjected to 5 sequential passages in total. (B) Infection of U87.CD4.CCR5 cells was initiated with VSV G-pseudotyped WT and W596L/K601D mutant viruses. The cells were trypsinized 24-h later to remove residually adsorbed viruses. The passaging procedure described in A was then followed. The results shown represent the mean RT activity ± standard deviation of triplicate samples. (C) Reversion pathways in WLKD-1 and WLKD-2 cultures. The env region was PCR amplified from proviral DNA isolated at days 10, 20, 30, 40 and 50, cloned into pΔKAD8env and sequenced. Upper case lettering connected by a bold horizontal line denotes a major evolutionary pathway, while lower case lettering connected via thin horizontal lines denotes a minor pathway. Lower case lettering only: low-frequency genotypes arising at the specified days.