Fig 6.

All of the SDs in the amino terminus of wt VEEV capsid protein play critical roles in packaging of viral genome and infectious virus release. (A) Schematic representation of the wt VEEV capsid protein and its variants with deleted amino-terminal subdomains. (B) A total of 5 × 10⁵ BHK-21 cells in 35-mm dishes were infected with VEEV/GFP and mutated genome-containing particles at an MOI of 20 inf.u/cell. Media were replaced at the indicated times postinfection, and the infectious titers were determined as described in Materials and Methods. (C) 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 harvested at 8 h postinfection, and the accumulation of virus-specific structural proteins was analyzed by Western blotting with VEEV-specific antibodies. (D) Quantitative analysis of VEEV structural proteins by Western blotting. The gel contains aliquots corresponding to 0.15 ml of the harvested media. (E) Comparative analysis of total viral RNA, genomic RNA, and actin RNA ratios in the released viral particles were determined by RT-qPCR. GE, genome equivalent.