FIG 4.

Expression of different GhV-F⁽⁺¹⁾ proteins in association with GhV-G and the resulting effect on pseudotyped virus formation and infectivity. Viral pseudotypes were generated on HEK293T cells transfected with plasmids encoding GhV-G and the GhV-F⁽⁺¹⁾ variants in a 1:1 ratio. Cells were simultaneously infected with a VSV-ΔG-rLuc virus complemented with VSV-G. At 24 h postinfection (hpi), pseudotype virus-containing media were clarified by low-speed centrifugation and then purified via ultracentrifugation through a 20% sucrose cushion. Purified GhV pseudotype particles (GhVpp) were resuspended in Opti-MEM, aliquoted, and stored at −80°C. (A and D) Expression of GhV-G and GhV-F′ or GhV-F⁽⁺¹⁾ variants in whole-cell lysates at 24 hpi (A) or in purified GhVpp (D). Primary anti-AU1 and anti-HA antibodies were used to detect AU1-tagged GhV-F and HA-tagged GhV-G, respectively. Fluorescent secondary antibodies were used for quantitative Western blotting, and the blots were imaged on a LI-COR Odyssey scanner. β-Tubulin is shown as a loading control. (B and E) The F₀ and F₁ bands from panels A and D were quantified using LI-COR Odyssey software, and expression levels for the GhV-F⁽⁺¹⁾ variants in whole-cell lysates (B) or GhVpp (E) were normalized to the expression of GhV-F′, which was set at 100%. (C and F) Cleavage efficiency of GhV-F⁽⁺¹⁾ variants in whole-cell lysates (C) or purified GhVpp (F) based on quantitative Western blot values. Data are presented as percent F cleavage, defined as [F₁/(F₀ + F₁)] × 100. GhV-F^(+T) was barely expressed in cell lysates and purified GhVpp, and thus, reporting cleavage efficiency values would be meaningless (asterisk). Results are representatives of 3 different experiments.