trans-complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication

Recently we described rescue of defective Kunjin virus (KUN) RNAs with smal l deletions in the methyltransferase and RNA polymerase motifs of the ns5 g ene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as h elper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now ex tended our previous observations and report successful trans-complementatio n of defective KUN RNAs with most of the ns5 gene deleted or substituted wi th a heterologous (dengue virus) ns5 sequence. Replication of full-length K UN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) n ucleotides in the ns5 gene was complemented efficiently in transfected repB HK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complem ented less efficiently, and RNA with an even larger deletion of 2,279 nucle otides (84%) was not complemented at all. Chimeric KUN genomic RNA containi ng 87% of the KUN ns5 gene replaced by the corresponding sequence of the de ngue virus type 2 ns5 gene was unable to replicate in normal BHK cells but was complemented in repBHK cells. These results demonstrate for the first t ime complementation of flavivirus RNAs with large deletions (as much as 75% ) in the RNA polymerase gene and establish that translation of most of the N-terminal half of NS5 is essential for complementation in trans. A model o f formation of the flavivirus replication complex implicating a possible ro le in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KU N and on reported data with other flaviviruses.