The relationship between N362, HIV-1 entry kinetics, and sensitivity to inhibition by neutralizing antibodies. Luciferase reporter viruses pseudotyped with a subset of PA-R5 Envs lacking N362 (NB24-C1, NB24-C2, NB24-C3, NB24-C4, NB25-C2, and NB25-C4) or with a subset of A-R5 Envs containing N362 (NB6-C2, NB6-C3, NB6-C4, NB7-C2, NB7-C4, NB8-C2 and NB8-C4) were produced and quantified as described in the Methods. The kinetics of HIV-1 entry by Env-pseudotyped luciferase reporter viruses was determined by time-of-addition studies with T-20, as described in the Methods (A). The results are expressed in minutes as the maximum delay time after addition of virus to JC53 target cells when addition of 50 μg per ml of T-20 can still completely inhibit HIV-1 entry (Max. delay of entry), which was determined by least squares regression analysis of time-of-addition curves. The sensitivity of Env-pseudotyped luciferase reporter viruses to neutralization by Env monoclonal antibodies IgG1b12 (B) or 2G12 (C), or by the polyclonal antibody HIV-Ig (D) was determined by calculation of IC50 and IC90 values by least squares analysis of neutralization curves, as described in the Methods. For sensitivity to neutralization by IgG1b12, four PA-R5 Envs (NB24-C1, NB24-C2, NB24-C3, NB24-C4) had IC50 and IC90 values > 50 μg per ml, indicating resistance. These Envs were assigned values of 50 μg per ml for the purpose of constructing panel (B). Box plots were constructed from mean values of duplicate experiments with each Env-pseudotyped luciferase reporter virus using Prism version 4.0c (GraphPad Software). Boxes represent upper and lower quartiles and median scores, and whiskers represent minimum and maximum values. The data shown are representative of 2 independent experiments. P values were calculated using a nonparametric Mann-Whitney U test, and values < 0.05 were considered statistically significant.