TRANSCOMPLEMENTATION OF FLAVIVIRUS RNA-POLYMERASE GENE NS5 BY USING KUNJIN VIRUS REPLICON-EXPRESSING BHK CELLS

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497-1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA p olymerase gene (NS5). A new infectious full-length KUN virus cDNA clon e, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway,, J. Virol. 68:4580-4588, 1994) and pos sessing similar to 10(5)-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of th e predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] bin ding site, producing FLdSAM) were introduced separately into FLSDX. Tr anscription and transfection of FLdGDD and FLdSAM RNAs into repBHK cel ls but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Ret ention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the p ossibility that recombination had occurred between the deleted defecti ve NS5 genes present in transfected RNAs and the functional NS5 gene p resent in the repBHK cells. An RNA with a point mutation in the GDD mo tif (FLGVD) was also complemented in transfected repBHK cells, and def ective virus was recovered by day 3 after transfection. However, in co ntrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days o r more) incubation of FLGVD RNA in normal BHK cells allowed recovery o f a virus in which the GVD mutation had reverted via a single base cha nge to the wild-type GDD sequence. Overall, these results represent th e first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.