Characterization of the nuclear localization signal in the avian sarcoma virus integrase

A sequence of 21 amino acids (aa) in the C-terminal region of the 286-aa av ian sarcoma virus (ASV) integrase (IN) protein has been shown previously to mediate nuclear localization of both IN and P-galactosidase (beta Gal) pro tein fused to it. This karyophilic sequence includes a high proportion of p rolines and residues with basic side chains. In this report, site-directed mutagenesis was used to introduce single aa substitutions of several of the se residues. Indirect immunofluorescence showed that IN-beta Gal fusion con structs with Ala substitutions for sequence constituents K206, P215, K225 o r R227 had lost the exclusive nuclear localization capability of the wild-t ype fusion. A fusion protein with the conservative substitution K206R retai ned the nuclear localization capacity. The site-specific substitutions that reduced karyophilic activity had no effect on the processing or joining ac tivities of IN in vitro. However, the introduction of three of the four Ala codon substitutions into viral DNA clones caused a significant delay in vi ral replication following transfection of cycling chicken embryo fibroblast s. These results are consistent with a possible role for ASV IN in nuclear targeting. (C) 1998 Elsevier Science B.V. All rights reserved.