Figure 3.

(A) Microinjection experiments confirm that the NS1 138–147 sequence is an NES. This NS1 sequence, its mutant (A substituted for the L at position 144), and the Rev NES were each fused to GST, and the resulting proteins were microinjected into the nuclei of COS cells. One hour after injection, the cells were fixed with formaldehyde, and the location of the GST fusions was determined by indirect immunofluorescence using rabbit anti-GST antibody and rhodamine-conjugated goat anti-rabbit antiserum. Mouse IgG, which was coinjected to verify the nuclear injection site, was detected using FITC-conjugated goat anti-mouse antibody. (B) Microinjection experiments demonstrate that the NS1 NES is inhibited by the adjacent 14 aa of the effector domain (amino acids 148–161). PCR was used to replace the NS1 NES in NS1(79–173) by Rev NES to produce NR(79–173). The two indicated GST-fusion proteins were microinjected into the nuclei of COS cells, which were analyzed by indirect immunofluorescence as described above.