FIG 3.

Nipah virus budding is significantly modulated upon the inhibition of endocytosis, recycling, and ESCRT function. (A) HEK293T cells were transfected with NiV F alone or with dominant-negative (DN) mutants of the following key vesicular trafficking factors: dynamin (endocytosis), Eps15 (clathrin-mediated endocytosis), VPS4A (ESCRT function), or Rab11 (recycling). Total cell lysates (CL) and virus-like particle (VLP) fractions were prepared 24 h after transfection and used for SDS-PAGE and Western blot analysis, whereas F cell surface expression (CSE) was measured using flow cytometry. (B) Using densitometry to assess the expression of the F protein in VLPs along with corresponding total cell lysate and CSE values, two indices were determined, VLP/CL and VLP/CSE. (C and D) As with panels A and B, a construct encoding G was coexpressed with each DN factor and the following CL, CSE, VLP, and index values elucidated. Similarly, coexpression with these constructs were tested for effects on M expression in cell lysates and VLPs but not at the cell surface due to it being a cytoplasmic protein. (E and F) Representative Western blots (E) and quantification (F) are shown. The results are representative of at least three experiments, with error bars indicating the standard error of the mean. One-way Student t tests were used to assess significant differences in budding efficiency indices compared to when they are cotransfected with pcDNA3.1, an empty vector (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).