Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes

Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for ex pression and delivery of heterologous genes. Replicon vector C20DX2Arep, co ntaining a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C v irus. The expression and proper processing of these genes upon transfection of BHK21. cells with the recombinant replicon RNAs were demonstrated by im munofluorescence, radioimmunoprecipitation, and appropriate reporter gene a ssays. Most of these recombinant KUN replicon RNAs were also successfully p ackaged into secreted virus-like particles (VLPs) by subsequent transfectio n with Semliki Forest virus replicon RNA expressing KUN structural genes. I nfection of BHK21 and Vero cells with these VLPs resulted in continuous rep lication of the recombinant replicon RNAs and prolonged expression of the c loned genes without any cytopathic effect. We also developed a replicon vec tor for generation of stable cell lines continuously expressing heterologou s genes by inserting an encephalomyelocarditis virus internal ribosomal ent ry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cel l lines persistently expressing GFP and CAT genes for up to 17 passages wer e established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of n oninfectious and noncytopathic vaccines as well as for gene therapy applica tions. (C) Academic Press.