Abstract:
:We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport. By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu. The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase. The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport. A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus. Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis. Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins. Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus. Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine. A double mutant at both positions was also blocked. We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect. These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins.
journal_name
Virologyjournal_title
Virologyauthors
Clever J,Kasamatsu Hdoi
10.1016/0042-6822(91)90472-nsubject
Has Abstractpub_date
1991-03-01 00:00:00pages
78-90issue
1eissn
0042-6822issn
1096-0341journal_volume
181pub_type
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