Activity analysis of N. benthamiana-expressed rAH.

(A) The gp120-binding capacity of rAH expressed in N. benthamiana was evaluated by gp120-ELISA using AH-expressing and control (uninfiltrated) leaf extracts prepared as described in Materials and Methods. Results of a representative sample obtained from a ∼2 g batch of leaf materials are shown. Data points are the means ± SD of triplicate analysis. This analysis was also used to determine the quantity of rAH in the extract based on a standard curve created by actinomycete-derived purified AH. (B) X-gal staining of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with: a, media alone; b, 1/20-diluted non-infiltrated control leaf sample; c, 1 µM actinomycete-derived AH; and d, 1/20-diluted rAH-expressing leaf sample (containing approximately 0.32 µM, or 4.0 µg/ml, of rAH, as determined by gp120-ELISA). (C) Quantitative analysis of syncytia formation by HeLa/Env/tat and HeLa/CD4/LacZ. The two cell lines were incubated for 18 h at 37°C with serially diluted control or rAH-expressing leaf samples. See Materials and Methods for the assay detail. Differences between the samples at each dilution point were evaluated by two-way analysis of variance followed by Bonferroni posttests (*** p<0.001).