Encapsidation of the Flavivirus Kunjin Replicon RNA by Using a Complementation System Providing Kunjin Virus Structural Proteins in trans

Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an ∼35-nm diameter, compared with ∼50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.