In vitro proteolytic processing of P4a, P4b, and P25K. 1 μl of TNT produced substrate either P4a (A), P4b (B), or P25K (C) was mixed with 5 μl of Hepes buffer and 14 μl of enzyme extracts, either from uninfected cells, or cells infected with ts16 at the permissive or non-permissive temperature. At the non-permissive temperature, plasmid borne I7L, either wild-type (pI7L) or mutant I7L (pI7LH241A) was transfected in as the source of enzyme. The reaction was incubated at 29°C for 3 hrs before being stopped by the addition of SDS sample buffer. Molecular weight is indicated on the left and the core protein precursor and product on the right. Lane 1 is substrate alone, lane 2 is substrate mixed with cellular extracts and lanes 3–5 are substrate mixed with the enzyme extract indicated.