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 approximately 100,000-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 approximately 5-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.

FIG. 1.

Schematic representation of the full-length and replicon KUN cDNA constructs with exchanged fragments. The filled box represents the sequence of the FLSDX clone; the open box represents the sequence of the pAKUN clone. SacII, SphI, BstBI, BssHII, AgeI, and SalI show restriction sites used in construction of plasmids by fragment exchange; numbers under the restriction sites represent corresponding nucleotide positions in the full-length KUN sequence (6, 10). In the designations of the full-length constructs generated by fragment exchange, first letters represent the name of the clone used as the vector backbone and are separated by a slash from the letters representing the name of the clone used as a source of the cloned fragment. Replicon constructs contain the puromycin acetyltransferase gene (PAC) and β-Gal gene (β-gal) cassette inserted in FLSDX(pro) in place of deleted structural genes (20). Amino acids shown in different recombinant constructs represent corresponding wild-type or mutated residues in NS2A (position 59) and NS3 (positions 518 and 557) genes identified in the original FLSDX and pAKUN constructs. Ile, isoleucine; Asn, asparagine; His, histidine; Pro, proline; Tyr, tyrosine; and Ser, serine.

FIG. 2.

Viral plaque morphology in BHK cells transfected with the KUN virion RNA (wild type [wt]) and with the indicated engineered RNAs (A) and infected with the corresponding recovered viruses (B). BHK cells were electroporated with indicated RNAs or infected with indicated recovered viruses and were assayed for plaque morphology as described in Materials and Methods. “ng RNA” under the panels in A shows the amount of transfected RNA in nanograms calculated from appropriate dilution of transfected cells in the corresponding dishes. Viral plaques in panel A were visualized at day 4 after RNA electroporation and in panel B at day 5 after infection with corresponding recovered viruses at 10⁻⁴ dilution.

FIG. 3.

IF analysis of BHK cells electroporated with mutated RNAs. One microgram of RNAs transcribed from indicated full-length KUN cDNA clones was electroporated into 2 × 10⁶ BHK21 cells, and 2 × 10⁵ cells were seeded on coverslips in 24-well plates. Twenty-four or 48 h later, cells were fixed with acetone and were stained with KUN anti-E antibodies as described in Materials and Methods.

FIG. 4.

Comparison of replication efficiencies of KUN replicon RNAs with mutations in NS2A and NS3. BHK21 cells were electroporated with the indicated mutated replicon RNAs. Forty-eight hours after electroporation, cells were either fixed by 4% formaldehyde-phosphate-buffered saline and were stained in situ with X-Gal (A) or lysed for a β-Gal assay (B). Total RNA was also isolated for analysis of accumulation of replicating RNA by Northern blot with ³²P-labeled probes specific for β-Gal (C, top row) or for β-actin (C, bottom row) nucleotide sequences. For panel C, ∼10 μg of total RNA was used for hybridization; arrows indicate positions in the gel of RNAs of 12.5 kb (C, top row) and 1.1 kb (C, bottom row) determined relative to migration in the same gel of the ethidium bromide-stained 1-kb ladder (Invitrogen). Numbering of corresponding panels (A), bars (B), and lanes (C) corresponds to cells transfected with repPACβ-gal RNA (1), repPACβ-gal/2Amut RNA (containing the Asn59 mutation) (2), repPACβ-gal/3mut(Pro557) RNA (3), repPACβ-gal/3mut RNA (with both His518 and Pro557 mutations) (4), and repPACβ-gal/3mut(His518) RNA (5).

FIG. 5.

Complementation of infectivity of KUN RNAs with NS2A mutations in repBHK cells. Electroporation of repBHK cells with RNAs and plaque assays were performed as described in Materials and Methods for normal BHK cells. “ng RNA” under the panels is defined as in the Fig. 1A legend. Viral plaques were stained at day 6 after electroporation.

FIG. 6.

Detection of packaged KUN replicon RNAs. Panels A and B represent selected fields of Vero cells stained with X-Gal at 48 h after infection with 100 μl of 1:100 dilution (panel A) or undiluted (panel B) culture fluids collected from BHK21 cells sequentially transfected with either repPACβ-gal (A) or repPACβ-gal/2Amut (B) RNAs, respectively, followed by transfection with SFV-MEC105 RNA.