Effects of E carboxyl tail mutations on E/N association and VLP production. (A-B) 293 T cells were transfected with HA-tagged E expression vector alone or in combination with M and/or N expression vectors. 74LL/AA indicates alanine substitutions for two leucine residues at the E carboxyl tail (74-LL-75). Δ76V denotes a deletion of the last carboxyl-terminal residue V76 from E. Supernatant and cells were harvested at 48 h post-transfection, prepared, and subjected to Western immunoblotting. E or N proteins from medium or cell samples were quantified by scanning the band densities from immunoblots. Ratios of HA-EΔ76V in media to those in cells were determined for each samples and normalized to that of HA-E in parallel experiment. The level of N-associated VLP (M + N) production is determined as described in the legend to Figure 1. (C) Removal of the last E carboxyl-terminal residue significantly affected E/N interaction. 293 T cells were co-transfected with HA-tagged wt or Δ76V and GST, GST-M or GST-N expression vectors. GST-N and GST-M have GST tags at the amino-terminals of the SARS-CoV N and M coding sequences, respectively. At 48 h post-transfection, cells and supernatants were collected and prepared for protein analysis. Aliquots of cell lysate samples were subjected to GST pull down analyses (right-hand panel).