FIG. 1.

Disruption of the predicted pseudoknot structure by the alanine-to-proline mutation at position 30 of the NS2A gene abolishes NS1′ production. (A) The frameshift motif and pseudoknot structure predicted for WT and A30P KUNV using pknotsRG software (21). The frameshift heptanucleotide and codon 30 of the NS2A gene are underlined, and the proposed interactions between bases in the predicted pseudoknot are shown as dashed lines. Altered nucleotides in codon 30 of the A30P mutant are highlighted. (B) Detection of NS1 and NS1′ in lysates of BHK, Vero, A549, and C6/36 cells 18 h after infection with WT or A30P KUNV viruses at an MOI of 5. Infected cells were pulsed with 50 μCi of [³⁵S]methionine in the methionine-free medium for 60 min, and then labeled medium was replaced with medium containing an excess of unlabeled methionine and cells were incubated for an additional 90 min. On completion of pulse-chase labeling, cells were lysed in 1% NP-40 lysis buffer and labeled NS1-containing proteins were immunoprecipitated with the anti-NS1 monoclonal antibody 4G4. Precipitated proteins were separated by SDS-PAGE and visualized on X-ray film. (C) Vero cells were transfected with DNA constructs coding for WT or A30P-mutated NS1-NS2A gene cassettes and incubated for 24 h prior to pulse-chase labeling and immunoprecipitation with 4G4 antibodies as in panel B.