FIG. 3.

Identification of the NES in VEEV capsid protein. (A) Sequence alignment of H68 peptides derived from different New World alphaviruses with a consensus NES sequence (NES), functional NES of cyclic-AMP (c-AMP)-dependent kinase inhibitor (PKI) (51) and supraphysiological NES (S1) (13). The sequence alignment was performed using ClustalW. Conservative hydrophobic amino acids in the NES are shown in red. Amino acids that are identical between different members of the New World alphaviruses are shaded in green. Asterisks indicate identical residues; colons indicate conserved substitutions; periods indicate semiconserved substitutions. WEEV, western equine encephalitis virus. (B) BHK-21 cells were coinfected with packaged replicons expressing VEEV capsid-GFP and 4×Tomato-3×NLS and treated with leptomycin B at 2 h postinfection as described in Materials and Methods. The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope. Scale bars, 20 μm. (C) (Top) Amino acid alignments of mutated H68-GFP fusions. Conserved hydrophobic amino acids are indicated in red, and introduced point mutations are shown in blue. (Bottom) Representative confocal images of cells expressing 4×Tomato-3×NLS and mutant proteins. Scale bars, 20 μm. (D) Box plot demonstrating the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS when expressed alone and the distribution of the same protein when coexpressed with mutant peptide-GFP. A small but statistically significant increase in nuclear-reporter accumulation was detected for single-amino-acid mutants. The double mutant no longer affected nuclear accumulation of 4×Tomato-3×NLS. (E) Box plot demonstrating the nuclear/cytoplasmic distributions of H68-GFP and its mutants. The increase in nuclear accumulation of the mutant-peptide-GFP fusions was correlated with their reduced efficiencies in nuclear import inhibition. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments).