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 small deletions in the methyltransferase and RNA polymerase motifs of the ns5 gene, using BHK cells stably expressing KUN replicon RNA (repBHK cells) as helper (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). We have now extended our previous observations and report successful trans-complementation of defective KUN RNAs with most of the ns5 gene deleted or substituted with a heterologous (dengue virus) ns5 sequence. Replication of full-length KUN RNAs with 3'-terminal deletions of 136 (5%), 933 (34%), and 1526 (56%) nucleotides in the ns5 gene was complemented efficiently in transfected repBHK cells. RNA with a larger deletion of 2,042 nucleotides (75%) was complemented less efficiently, and RNA with an even larger deletion of 2,279 nucleotides (84%) was not complemented at all. Chimeric KUN genomic RNA containing 87% of the KUN ns5 gene replaced by the corresponding sequence of the dengue 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 time 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 of formation of the flavivirus replication complex implicating a possible role in RNA replication of conserved coding sequences in the N-terminal half of NS5 is proposed based on the complementation and earlier results with KUN and on reported data with other flaviviruses.
Khromykh
A A
AA
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia. a.khromykh@mailbox.uq
Sedlak
P L
PL
Westaway
E G
EG
D015152:Blotting, Northern; D002460:Cell Line; D003673:Defective Viruses / Q000235:genetics; D018349:Encephalitis Viruses, Japanese / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D005816:Genetic Complementation Test; D008954:Models, Biological; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication
C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins