(A) Relationship between sCD4 sensitivity, CD4-Ig binding and infection of primary macrophages (mΦ) of DE86 and DG08 viruses. Values above the bars indicate fold increase in sCD4 sensitivity of evolving viruses compared to early viruses, and the vertical dashed line indicates the time of coreceptor switching. sgp120 binding to CD4-Ig was normalized to that of sgp120 binding to polyclonal serum from HIV-1 infected individuals. Infectivity in mΦ that express low levels of CD4 was expressed as a ratio of infectivity in autologous PBMCs that express high levels of CD4 and CCR5. The shaded area highlights the time prior and during coreceptor switch. For sgp120 CD4-Ig binding, data are the means and standard deviations from at least two independent experiments. For infection of macrophages, data are representative of at least 3 independent experiments (error bars, s.d.). * indicates statistically significant differences between the early and the evolving R5 viruses. (B) Changes in neutralization sensitivity of R5 viruses evolving over time in macaques DE86 and DG08. Susceptibility of R5 pseudoviruses to neutralization with IgG1b12, 447-52D and T20 was determined. The vertical dashed line indicates the time of coreceptor switching, and the shaded area designates the period of marked envelope conformational changes. Data are representative of at least two independent experiments (error bars, s.d.). * above the bars indicate IC50 values that are statistically different between the acute and the evolving R5 viruses, P<0.05 (Mann-Whitney U test).