cis- and trans-acting elements in flavivirus RNA replication

Most of the seven flavivirus nonstructural proteins (NS1 to NS5) encoded in the distal two-thirds of the RNA positive-sense genome are believed to be essential components of RNA replication complexes. To explore the functiona l relationships of these components in RNA replication, we used trans-compl ementation analysis of full-length infectious RNAs of Kunjin (KUN) virus wi th a range of lethal in-frame deletions in the nonstructural coding region, using as helper a repBHK cell line stably producing functional replication complexes from KUN replicon RNA. Recently we showed that replication of KU N RNAs with large carboxy-terminal deletions including the entire RNA polym erase region in the NS5 gene, representing 34 to 75% of the NS5 coding cont ent, could be complemented after transfection into repBHK cells. In this st udy we have demonstrated that KUN RNAs with deletions of 84 to 97% of the N S1 gene, or of 13 to 63% of the NS3 gene including the entire helicase regi on, were also complemented in repBHK cells with variable efficiencies. In c ontrast, KUN RNAs,vith deletions in any of the other four nonstructural gen es NS2A, NS2B, NS4A, and NS4B were not complemented. We have also demonstra ted successful trans complementation of KUN RNAs containing either combined double deletions in the NS1 and NS5 genes or triple deletions in the NS1, NS3, and NS5 genes comprising as much as 38% of the entire nonstructural co ding content. Based on these and our previous complementation results, we h ave generated a map of cis- and trans-acting elements in RNA replication fo r the nonstructural coding region of the flavivirus genome. These results a re discussed in the context of our model on formation and composition of th e flavivirus replication complex, and we suggest molecular mechanisms by wh ich functions of some defective components of the replication complex can b e complemented by their wild-type counterparts expressed from another (help er) RNA molecule.