Fig. 1

Phenotypic comparison of transmitted founder and matched chronic infection Envelopes. Env sampled from a participant at 5 (transmitted founder; TF) and 173 (chronic infection; CI) weeks post-infection were expressed in HEK293T cells. a The sequences of the two clones (C15 and C16) were aligned in Bioedit and PNGs are indicated with asterisk. Gp120 sequence is shown starting from the first potential N-glycan site (PNG) at position 88 (HXB2 numbering) with variable loops indicated. b Env expression was determined in the presence and absence of pSG3Δenv by Western blotting. β-actin, a protein loading control, gp160 and gp120 are indicated with arrows. A representative of three independent experiments is shown. c Gp140 was digested with EndoH and PNGaseF and the molecular weight (MW) was compared to that of undigested gp140 using Western blotting. Mannosylation (%) was determined using the equation: [(Untreated – EndoH-treated)/(Untreated) − (PNGaseF-treated)] × 100. The average of two biological repeats with standard error of the mean (SEM) are indicated. Statistical analysis was done using One-way Anova. Pseudovirus (PSV) was compared for their ability to d infect TZM-bl cells, e bind to Raji-DC-SIGN cells and trans-infect TZM-bl cells after capture by f Raji-DC-SIGN or g MDDCs. The average relative light units (RLU) of four independent entry efficiency experiments are indicated. DC-SIGN binding of three independent experiments was calculated relative to PSV input (%) and trans-infection of four biological experiments are indicated relative to the entry efficiency of equivalent PSV (%). Error bars represent SEM and statistical analysis was done using Mann Whitney U test. h TF and i CI Env potential N-glycan sites (PNGs) at N241, N262, N448, N386 and N392 (HXB2 numbering) were deleted by site-directed mutagenesis and compared to WT for their ability to infect TZM-bl cells, bind to DC-SIGN, and trans-infect TZM-bl cells. Bars indicate the mean of two independent experiments with SEM. Statistical analysis was done using One-way Anova and Bonferroni’s post-test. PSVs generated using pSG3∆Env and empty vector was used as a negative control (Ctrl). For all statistical analysis, *, ** and *** indicate p values < 0.05, p < 0.01 and p < 0.001, respectively relative to WT, ns was not significant