Efficient trans-complementation of the flavivirus Kunjin NS5 protein but not of the NS1 protein requires its coexpression with other components of the viral replicase

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FL dGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by usi ng a BHK cell line, repBHK that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A . Khromykh M. T, Kenney, and E. G. Westaway, J, Virol, 72:7270-7279, 1998), In order to identify whether this complementation of FLdGDD RNA was provid ed by the wild-type NS5 protein alone or with the help of other nonstructur al (NS) proteins also expressed in repBHK cells, we generated BHK cell Line s stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 pol yprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon, Western blot analysis with anti-NS 5 antibodies showed that the level of production of NS5 was significantly h igher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficie nt in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fol d less of the secreted complemented virus. In contrast, efficient complemen tation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein fr om the Sindbis replicon vector and in repBHK cells, with both cell lines ex pressing similar amounts of NS1 protein, These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicas e possesses much higher functional (trans-complementing) activity than indi vidually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formati on of the flavivirus RC largely based on previous ultrastructural and bioch emical analyses of KUN replication.