FIG 6.

A single polymorphism, T544I, in GP2 accounts for the differential ability of EBOV2014-GP and EBOV1976-GP to drive entry into nonhuman primate cells. (A) The sequences spanning the border between GP1 and GP2 were aligned for the wt and mutant GPs analyzed. Conserved amino acid residues are marked with asterisks, while residues that differ between two GPs are indicated by bold, red letters. Amino acid residues of the internal fusion loop (IFL) are highlighted by a gray box. (B) All complete EBOV-GP sequences available at NCBI database that were not linked to the West African epidemic (n = 66) were analyzed for the amino acid present at position 544. The relative distribution of threonine (orange) and isoleucine (green) is shown. (C) Rhabdoviral pseudotypes harboring wild-type (wt) EBOV1976-GP, EBOV1976-GP (I544T) (both blue), wt EBOV2014-GP, or EBOV2014-GP (T544I) (both red) were inoculated onto COS-7 cells. Pseudotypes that contained no glycoprotein (pCAGGS, white) or VSV-G (gray) served as negative and positive controls, respectively. At 18 h postinoculation, transduction efficiency was quantified by measuring firefly luciferase activity in cell lysates. The average of three independent experiments (with separate pseudotype preparations) is shown. Results were normalized against transduction mediated by EBOV1976-GP wt (set as one) and are shown as x-fold changes. Error bars indicate the standard error of the mean. (D) EBOV-based VLPs harboring either no glycoprotein (white, negative control), wt EBOV1976-GP, EBOV1976-GP (I544T), wt EBOV2014-GP, or EBOV2014-GP (T544I) were inoculated onto COS-7 cells. At 3 h postinoculation, the firefly luciferase activity in cell lysates was quantified, corrected for background activity (measured in cells inoculated with pseudotypes not bearing a glycoprotein), and normalized against luciferase activity of VLPs used for inoculation (input factor). The results of a representative experiment carried out with quadruplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in two separate experiments performed with independent VLP preparations. Paired (pseudotypes) and unpaired (VLPs) two-tailed Student t tests were used to assess statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance).