Effect of virus replication, CD4 binding affinity, and coreceptor tropism on PDC IFN-α induction. (a to c) Transfection of decreasing amounts of pBRNL4-3 DNA resulted in parallel decreases of gp120 expression levels (a), p24 antigen production (b), and IFN-α (c) induction in PDC cocultures. Data represent three to six separate experiments. Statistical analysis was performed using one-way analysis of variance for trend analysis. Controls included unstimulated PDC (mock), calf thymus DNA (carrier) substituting for the total amount of transfected DNA, and pBRdeltaNL4-3, deleted for all sequences coding for retroviral genes. (d) Expression of gp120 in transfected 293T cells using pBRNL4-3 mutants with either completely abolished (D368G) or reduced (D368N) CD4 affinity. Controls were pBRNL4-3-transfected 293T cells incubated with the fluorescein isothiocyanate-conjugated secondary antibody but without 2G12. One representative FACS histogram out of three separate experiments is shown. (e) IFN-α induction by transfected 293T cells using PDC obtained from four different donors. Data are shown as means and standard errors normalized for gp120 expression. (f) CD4 binding affinities of the respective viruses, determined in three separate experiments. (g and h) Linear regression analysis of the number of positively charged amino acids in the V3 loop (Table 2) (g) and the mean CD4 binding affinity of 17 HIV-1 V3 loop and Env recombinants with respect to the mean induction of IFN-α production (h), normalized for gp120 expression in five separate experiments (n.s., not significant). OD, optical density.

Effect of CD4 and CXCR4 on the attachment of HIV-1 to PDC. (a and b) Time-lapse experiments evaluating the attachment of autofluorescing HIV particles (pNLC4-3^EGFP) (33) to PDC after different incubation periods (minutes) using FACS analysis (data are representative of four independent experiments) (a) and immunofluorescence microscopy (b). (c) Blocking of the attachment of pNLC4-3^EGFP to PDC using monoclonal antibodies to CD4 (Leu3a) compared to an isotype control (mIgG1). (d and e) Effect of anti-CD4 (d) and anti-CXCR4 (e) antibodies on the attachment of pNLC4-3^EGFP to PDC, shown as means and standard errors of six and three separate experiments, respectively, comparing the mean fluorescence intensities (MFI) after 10 minutes of incubation. n.s., not significant.