In vitro analysis of the inhibition of HIV-1 replication by IFNα and IFNβ. (A) Comparison of the inhibition of HIV (W6BC) replication when cells were treated with IFNα or IFNβ for 4 hours prior to infection (open bars) or were treated with IFNα or IFNβ both prior to infection and throughout the subsequent viral replication period (filled bars). Viral replication was assessed by measurement of supernatant p24 levels on day 7 post-infection. The results shown are the mean p24 values from 5 replicate wells treated with the indicated concentrations of IFN, expressed as a % of p24 values from cells that were not IFN-treated. Error bars represent 1 standard deviation above the mean. (B) Example of data from assays performed to assess the inhibition of a representative primary HIV-1 isolate (generated from plasma cryopreserved during acute infection from subject MM38) by IFNα and IFNβ. Inhibition of virus replication by each IFN subtype was assessed in two independent assays using mixed PBMCs derived from different groups of donors (black and grey bars). The results shown are the mean p24 values from 4 replicate wells treated with the indicated concentrations of IFN, expressed as a % of p24 values from cells that were not IFN-treated. Error bars represent 1 standard deviation above the mean. (C) Calculation of Vres and IC50 values from a representative IFN inhibition assay (the first IFNβ assay performed on the acute time-point virus isolate from subject MM38 in B). The level of viral replication in the presence of IFNβ (mean supernatant p24 concentration, expressed as a percentage of the mean p24 concentration in the absence of IFN) is plotted against the IFNβ concentration and a curve fitted to the data by non-linear regression using a least squares method. The Vres value (level of virus replication observed in the presence of maximally-suppressive IFN concentrations) is indicated by the dotted line. The IC50 value (IFN concentration required to produce half-maximal inhibition of viral replication i.e. midway between 100% replication and Vres) is read off from the inhibition curve as indicated by the dashed line.