Stability of NS2 proteins expressed by subgenomic VEE replicons.

<p>(A) At the top is shown the organization of subgenomic VEE replicons expressing the H77, JFH1, and chimeric HJ2 (“H/J”) NS2 protein. Arrows indicate the VEE subgenomic promoter, while “Pac” indicates the puromycin resistance gene. Shown below are the different NS2 proteins expressed by this series of replicons; each contains either an N- or C-terminal Flag (or Myc) tag. (B) Immunoblot detection of NS2 protein using anti-Flag (left panel) or anti-Myc (right panel) antibodies. With the exception of sig-H77-NS2-Flag, which has a Flag tag at the C-terminal end of the NS2 protein, all of the other NS2 proteins have a Flag or Myc tag at the N-terminus. (C) Treatment with the proteasome inhibitor MG132 enhances the abundance of H77 NS2 (both wt and to a lesser extent the S168G mutant, “SG”) but not JFH1 NS2 expressed by VEE replicon cell lines.