SIMULTANEOUS ACCUMULATION OF MULTIPLE VIRAL COAT PROTEINS FROM A TEV-NIA BASED EXPRESSION VECTOR

We previously described an expression cassette that relies on the toba cco etch virus (TEV) nuclear inclusion a (NIa) protease and leads to t he coordinated accumulation of multiple proteins through self processi ng of a polyprotein [21]. However, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. Studies were conducted to evaluate whether the disruption of NIa nucl ear localization would affect the levels of proteins produced via the cassette. Modifications comprised either removal of its nuclear locali zation signals (NLSs), removal of the VPg domain (which includes the N LSs), and fusion to the 6 kDa protein, previously demonstrated to be a viral cytoplasmic anchor [28]. In in vitro translation reactions and in vivo protoplast experiments the modified NIa retained sequence-spec ific proteolysis. Moreover, the removal of the NLSs correlated with an increase in GUS reporter accumulation. The modified cassette, pPRO 10 , led to the synthesis of up to three viral coat protein (CPs) in addi tion to NIa. However, the accumulation of proteins in protoplasts depe nded upon the position of the CP coding sequence within the cassette a s well as on the stability of the protein.