SUBGENOMIC REPLICONS OF THE FLAVIVIRUS KUNJIN - CONSTRUCTION AND APPLICATIONS

Several Kunjin virus (KUN) subgenomic replicons containing large delet ions in the structural region (C-prM-E) and in the 3' untranslated reg ion (3'UTR) of the genome have been constructed. Replicon RNA Delta ME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene , inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (f rom nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated e fficiently in electroporated BHK21 cells. A further deletion from C20r ep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Re plicon Delta ME/76 with a deletion of 76 nucleotides in the 3'UTR of D elta ME RNA (nucleotides 10423 to 10498) replicated efficiently, where as replicon Delta ME/352 with a larger deletion of 352 nucleotides (nu cleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the po ssibility of using a reporter gene assay to monitor synthesis of the p ositive strand and the negative strand of KUN RNA, we inserted a chlor amphenicol acetyltransferase (CAT) gene into the 3'UTR of Delta ME/76 RNA under control of the internal ribosomal entry site (IRES) of encep halomyelocarditis virus RNA in both plus (Delta ME/76CAT[+])- and minu s (Delta ME/76CAT[-])-sense orientations. Although insertion of the IR ES-CAT cassette in the plus-sense orientation resulted in a significan t (10- to 20-fold) reduction of RNA replication compared to that of th e parental Delta ME/76 RNA, CAT expression was readily detected in ele ctroporated BHK cells. No CAT expression was detected after electropor ation of RNA containing the IRES-CAT cassette inserted in the minus-se nse orientation despite its apparently more efficient replication (sim ilar to that of Delta ME/76 RNA); this result indicated that KUN negat ive-strand RNA was probably not released from its template after synth esis. Replacement of the CAT gene in the Delta ME/76CAT(+) RNA with th e neomycin gene (Neo) enabled selection and recovery of a BHK cell cul ture in which the majority of cells were continuously expressing the r eplicon RNA for 41 days (nine passages) without apparent cytopathic ef fect. The constructed KUN replicons should provide valuable tools to s tudy flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.