FIG 4.

A region including a membrane-proximal polybasic cluster mildly modulates budding of F. (A) Schematic for the cytoplasmic tail of NiV F designating the region being alanine screened. (B) HEK293T cells were transfected for 24 h with an empty vector, wild-type NiV F, one of the five alanine mutants of region T1, or the NT double mutant. Both cell lysates and VLPs were harvested and run in SDS-PAGE followed by Western blot analysis. (C) Densitometry was used to quantify band intensity for cell lysates and VLPs. In addition, flow cytometric analyses were used to evaluate the levels of CSE for each of the mutants relative to the wild-type F. (D) The cell lysate and VLP band intensities were used to calculate budding indices and all were normalized to wild-type F. A similar index is also shown using values for VLP intensity and corresponding CSE values. Error bars designate values for standard errors of the mean. Three or more independent experiments were used for each ratio, and the statistical significance was evaluated with one-sample t tests.