Fig 6.
NS3p is proteolytically active. BHK-21 cells were infected with vaccinia virus MVA/T7 pol and transfected with the NS4-5 substrate or cotransfected with plasmids encoding enzymes and substrates. (A) Expression of the enzymes (NS3, NS3-4A, NS2-3C1512A, NS3p, and NS3p-4A) is documented by Western blotting using moAb GL3p1. Autocatalytic NS3-4A cleavage removes the 4A moiety of NS3-4A and NS3p-4A. Introduction of C1512A abrogates the activity of autocatalytic NS2 protease, to prevent NS2-3 cleavage. (B) Enzymatic activity of the constructs was analyzed with a moAb GR5B1. The positions of NS2-3, NS3, NS3p, NS4-5, and mature NS5B are indicated at the right. A scheme of NS3 enzyme constructs, NS4-5 substrate, and NS5B product is shown at the right. Molecular mass standards are shown on the left.