Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

Abstract

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...

Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

“…It was reported that NS2A binds with high specificity to the 3' untranslated region (UTR) of viral genomic RNA and to other components of the replication complex (Mackenzie et al, 1998). In addition, NS2A is considered to play roles in modulating the host-antiviral interferon response (Liu et al, 2004;Liu et al, 2006;Liu et al, 2005;Munoz-Jordan et al, 2003) and assembly/ secretion processes of virus particles (Kummerer and Rice, 2002;Leung et al, 2008;Liu et al, 2003). Although the exact membrane topology of NS2A is yet to be determined, NS2A of OHFV has been predicted to span the membrane of the endoplasmic reticulum five times by several transmembrane domain prediction programs (TMHMM (Krogh et al, 2001), and TMpred (Hofmann and Stoffel, 1993)).…”

IS-98-ST1 West Nile virus derived from an infectious cDNA clone retains neuroinvasiveness and neurovirulence properties of the original virus

“…Different approaches have thus been used to improve the stability of full-length flavivirus cDNA clones [3]. Introns have been inserted to interrupt genes encoding products that may be toxic in bacteria [8,36], and relatively permissive E.coli strains have been identified. Full-length flavivirus cDNA from NY99, B956 and Kunjin WNV strains have previously been cloned in plasmids amplified in the HB101 E.coli strain [3,8,26].…”

“…Introns have been inserted to interrupt genes encoding products that may be toxic in bacteria [8,36], and relatively permissive E.coli strains have been identified. Full-length flavivirus cDNA from NY99, B956 and Kunjin WNV strains have previously been cloned in plasmids amplified in the HB101 E.coli strain [3,8,26]. In the present study, we employed a plasmid cloning strategy to construct an infectious clone of the IS-98-ST1 WNV strain (WNV-IC).…”

“…Selection of the appropriate plasmid is critical [3,37]. The pBR322 plasmid was chosen because it had been used successfully to generate West Nile infectious clones in the past [3,8,26]. Plasmids encoding flavivirus sequences downstream of cytomegalovirus (CMV) or lac promoters displayed high instability due to expression of products that were toxic for E.coli [38].…”

“…Plasmids encoding flavivirus sequences downstream of cytomegalovirus (CMV) or lac promoters displayed high instability due to expression of products that were toxic for E.coli [38]. To avoid such problems we selected the promoter for the bacteriophage SP6 RNA polymerase for in vitro transcription of viral RNA [8,26,39,40]. An additional G nucleotide was inserted downstream of the promoter to ensure more efficient transcription initiation [26].…”