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.