FIG. 2.
Mapping of causal mutations in the variant replicons. (A and B) Schematic illustrations of the cloning strategy used to map the replicon variants. Depicted are the neo-containing SIN and SFV replicons, with corresponding RT-PCR-amplified fragments generated from the Neo-resistant BHK pools. Also shown are nsP coding regions and the restriction sites used in fragment substitutions. nt., nucleotide. The ability of each fragment substitution to efficiently confer Neo resistance (+) compared to the parental vectors (−) is indicated; “nt” denotes fragments not tested. (C) Sequence alignments of the nsP2 regions in which the mutations were located are shown for several alphaviruses. Bold characters indicate amino acid residues where mutations were found; the change is indicated above the alignment for the SIN-derived variants and below the alignment for the SFV-derived variants. In variant SF1B, Δ indicates the deletion of amino acid D469. Since the length of nsP2 varies between SIN and SFV, codon numbering is indicated for each virus. White boxes highlight identical residues among all of the alphaviruses aligned; gray boxes highlight conservative changes. Images were processed with Adobe Photoshop software.