Buoyant density and electron microscopy analysis of the mutant PFV particles. 293T cells were co-transfected with either pcoPG4 (wt), pcoPG4 μgR (μgR), or pcoPG4 GR R/A (GR R/A) in combination with pcoPP, pcoPE and puc2MD9 to yield the respective wt and mutant particles. (A) Concentrated virus supernatant was loaded onto an iodixanol step gradient ranging from 15 to 40%. After centrifugation at 197,000 g for 3 h the gradient was split into 17 fractions and their density determined by refractometry measurements (right ordinate) and their Gag content examined by Western blot analysis (left ordinate). The highest Gag signal intensity in each gradient was set to 100%. (B) Western blot analysis of the Gag content in individual gradient fractions (1-17), pelleted virus particles (L) and cell lysates of transfected 293T cells (CL) using polyclonal antibodies specific for PFV Gag (α-Gag) (see above). (C) Cryo-electron microscopy analysis of wild type PFV (left) and of GR R/A mutant (right) particles. The virus particles were concentrated via a 20% sucrose gradient centrifugation step followed by size exclusion column filtration. Black arrowheads indicate regular Gag assemblies in the wild type virus and white arrows mark putatively aberrant Gag assemblies in the GR R/A mutant. Scale bar: 60 nm.