A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ϳ100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ϳ5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.Kunjin virus (KUN) is an Australian flavivirus closely related to other members of the Japanese encephalitis virus subgroup. The KUN genome consists of single-stranded RNA of positive polarity comprising 11,022 nucleotides (10), with one long open reading frame coding 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (6). The gene order of KUN genome RNA is 5Ј-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3Ј.Generation of the flavivirus full-length cDNA clones has been hampered by their apparent instability in Escherichia coli, leading to extensive mutagenesis of cDNA sequences during preparation of plasmid DNAs (25). These mutations usually result...

The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in ϳ15-to 50-fold more efficient establishment of persistent replication in hamster ( (29), with one long open reading frame coding for 3,433 amino acids in three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (7). The gene order of KUN genome RNA is 5Ј-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3Ј. We previously constructed the first flavivirus replicons based on KUN cDNA by deleting the majority of the genomic region including structural genes (30) and used them extensively for the development of a gene expression system (2,19,28,30,44,45). KUN replicons were also used extensively in RNA replication and complementation studies (23)(24)(25)(26)(27)32) that contributed substantially to generating a comprehensive model for the formation and operation of the flavivirus RNA replication complex (47,48).The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses was shown to lead to the generation of adaptive mutations in nonstructural genes that either decreased (alphavirus replicons) or enhanced (hepatitis C replicons) RNA replication efficiency (1,4,13,14,34,39,41,50). Some of these adaptive mutations (e.g., in the NS5A protein of the hepatitis C replicon) were shown to increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-␣/␤) (41), while others (e.g., in the nsP2 protein of Sindbis virus) were shown to lead to more efficient IFN production (14). IFN response is the first line of cell defense against viral infections, and a majority of viruses have developed various strategies to overcome it, by either inhibiting IFN production or blocking IFN signaling (15,22,35,43). Recent studies of dengue virus have indicated that flaviviruses may interfere with early steps of IFN signaling and have implied roles for the small nonstructural proteins NS2A, NS4A, and NS4B in this process (37).In this study, we describe the identification of adaptive mutations in KUN replicon RNA that confirm an advantage in establishing persistent replication in a number of different cell lines. We also demonstrate that induction of IFN-␤ promoter-* Correspondi...