title: 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.
abstract: Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using 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 provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation 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 from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually 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 formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.
authors.affiliation: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia. a.khromykh@mailbox.up.edu.au
authors.forename: A A
authors.initials: AA
authors.lastname: Khromykh
authors.forename: P L
authors.initials: PL
authors.lastname: Sedlak
authors.forename: K J
authors.initials: KJ
authors.lastname: Guyatt
authors.forename: R A
authors.initials: RA
authors.lastname: Hall
authors.forename: E G
authors.initials: EG
authors.lastname: Westaway
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004279:DNA, Viral; D018349:Encephalitis Viruses, Japanese / Q000235:genetics*; D015870:Gene Expression; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D016296:Mutagenesis; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D011817:Rabbits; D012093:Replicon; D012845:Sindbis Virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics*
chemical_list: D004279:DNA, Viral; C100315:NS1 protein, Flavivirus; C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase