trans -Complementation of Flavivirus RNA Polymerase Gene NS5 by Using Kunjin Virus Replicon-Expressing BHK Cells

Abstract

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497–1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway, J. Virol. 68:4580–4588, 1994) and possessing ∼105-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLdGDD and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FLGVD) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days or more) incubation of FLGVD RNA in normal BHK cells allowed recovery of a virus in which theGVD mutation had reverted via a single base change to the wild-type GDD sequence. Overall, these results represent the first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.

The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

“…The multiple points of regulation present a formidable theoretical barrier to extensive deletions or insertions in polyproteins. Several groups working with positive-stranded RNA viruses have reported deletions of portions of protein domains or the generation of viruses whose extensive defects in replication require rescue by replicons or helper viruses (16,26,29,32,34,38,39,44,48). However, for animal positive-stranded RNA viruses, we have identified no published reports of autonomously replicating mutant viruses with deletions of complete mature protein domains within polyproteins.…”

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication

“…While ideal, trans-complementation using protein expression has not been successful for coronaviruses. In fact, efficient complementation for other positive-strand RNA viruses has required the development of replicon systems to facilitate viral replication complex formation (Appel et al, 2005;Grassmann et al, 2001;Khromykh et al, 1998;Lindenbach and Rice, 1997;Liu et al, 2002). Despite these limitations, this is the first report of detailed mutagenesis of an MHV gene 1 nsp-coding region within the context of a virus.…”

Successful Propagation of Flavivirus Infectious cDNAs by a Novel Method To Reduce the Cryptic Bacterial Promoter Activity of Virus Genomes

“…A third approach involves the use of medium-or low-copy-number plasmids to stabilize full-length flavivirus cDNAs. This strategy has been employed with different strains of DENV2 cDNAs (23,36,51,71), West Nile virus cDNA (55,66), Kunjin virus cDNA (34,35), and TBEV cDNA (43). Two other modified methods have been derived from this strategy to facilitate the construction of flavivirus cDNAs.…”