SEMLIKI FOREST VIRUS EXPRESSION SYSTEM - PRODUCTION OF CONDITIONALLY INFECTIOUS RECOMBINANT PARTICLES

In the recently developed Semliki Forest virus (SFV) DNA expression sy stem, recombinant RNA encoding the viral replicase, and helper RNA mol ecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence need ed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication -proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal pr oteolytic processing to activate viral entry functions. Hence, the rec ombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We the refore conclude that this conditional expression system meets extremel y stringent biosafety requirements.