The replication of tick-borne encephalitis virus (TBEV), like that of all 
flaviviruses, is absolutely dependent on proteolytic processing. Production of 
the mature proteins C and prM from their common precursor requires the activity 
of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and 
the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, 
we have shown in cell culture that the cleavage of protein C and the subsequent 
production of TBEV particles can be made dependent on the activity of the 
foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 
protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we 
developed an in vitro cleavage assay to assess the two cleavage reactions 
performed on the C-prM precursor. Accordingly, a recombinant modular 
NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain 
of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This 
enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. 
However, cleavage was only specific when protein synthesis was performed in the 
presence of canine pancreatic microsomal membranes and required the prevention 
of signal peptidase I (SPaseI) activity by lengthening the h-region of the 
signal peptide. The presence of membranes allowed the concentration of 
NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) 
cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing 
in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at 
the C-prM junction. This system shows that processing at the C-terminus of 
protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow 
the examination of how the membrane topology of protein C affects both SPaseI 
and NS2B/3(pro) processing.