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Expression and fusogenicity of mutant BLV envelopes.

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posted on 2013-02-20, 20:48 authored by Daniel Lamb, Alexander W. Schüttelkopf, Daan M. F. van Aalten, David W. Brighty

A panel of 10 mutants in BLV envelope was generated using site-directed mutagenesis of the pCMV-BLVenv-RRE vector. The envelopes (or empty pCDNA3.1 for the mock sample) were transfected with pRSV-Rev into HeLa cells, and the cells were split into three aliquots, one sample was lysed for Western analysis, another stained and examined by flow cytometry, and the third used as effector cells in syncytium formation assays with non-transfected HeLa cells as target cells. (A) Western blot probed for SU (upper panel) and actin (lower panel). Upper band is the Env precursor, gp72, lower bands gp51. (B) Flow cytometry analysis using the same primary antibody as in (A), detected with FITC-labelled anti-mouse IgG. Solid histograms represent mock transfected cells, shown alone in the first panel, and open histograms represent the envelope transfected cells. (C) Fusogenicity of the mutant envelopes. Fusogenic index represents the number of nuclei in a low-power light microscope field present in syncytia as a percentage of the total nuclei in the field (Data represent the means from triplicate assays ±SD).