Fig 3.

The entire amino-terminal domain of VEEV capsid protein is dispensable for VLP formation. (A) Schematic representation of the VEEV/Cm genome and mutants with multiple subdomains deleted. (B) Analysis of viral particles released from cells infected at an MOI of 20 inf. u/cell at 20 h postinfection. Particles were collected, concentrated, pelleted, and analyzed by SDS-PAGE as described in Materials and Methods. The gel was stained by Coomassie brilliant blue, and aliquots corresponding to 2.25 ml of the harvested media are presented. (C) Quantitative analysis of VEEV structural proteins by Western blotting of the samples presented in panel B. The gel contains aliquots corresponding to 0.15 ml of the harvested media. (D) Analysis of viral protein expression in BHK-21 cells infected with the indicated mutants. Cells were infected at an MOI of 20 inf.u/cell and harvested at 8 h postinfection, and the accumulation of virus-specific structural proteins was analyzed by Western blotting with VEEV-specific antibodies. (E) Negative staining and EM analysis of concentrated VLPs released from cells infected with different mutants (see Materials and Methods for details). The mean sizes of the particles and the standard deviations are presented. Scale bars, 100 nm.