Biochemical characteristics of functional domains using feline foamy virus integrase mutants

Abstract

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

Fig. 1.. Structure and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the integrase deletion mutant. (A) Primary structures. The feline foamy virus (FFV) integrase is divided into N-terminal, central, and C-terminal domains. We named N: N-terminal domain (NTD); T: central domain; C: C-terminal domain; NT: N-terminal domain + central domain; TC: central domain + C-terminal domain. Substitutions in the FFV integrase point mutants are marked with an X. (B) SDS-polyacrylamide gel electrophoresis of FFV deletion mutant integrases. Purified wild-type and deletion mutant integrases were separated by 16.5% SDS-polyacrylamide gel electrophoresis, and the gel was stained with Coomassie Blue (low molecular weight standards; 14, 21.5, 31, 45, and 66.2 kDa from the bottom of the gel).

Fig. 2.. In vitro complementation assays for 3'-end processing, strand transfer, and disintegration activities of the feline foamy virus (FFV) integrase deletion and point mutants. (A) 3'-end processing reactions of the deletion mutant integrases on an FFV U5 substrate. (B) Strand transfer activity of the deletion mutant integrases on an FFV U5 substrate. The strand transfer products (integration) appeared as bands larger than the 18mer. (C) Disintegration activities of the deletion mutant integrases on the FFV U5 substrate. (D, E) Complementation of 3'-end processing activities between deletion and point mutants. The 3'-end processing of D107A, D164H, E200A, E200D, and E200H data are not shown. (F) Complementation of disintegration activity between the deletion and D107A point mutant. (G) Complementation of disintegration activity between the deletion and D107H point mutants. (H) Complementation for disintegration activity between deletion and E200A point mutants. Disintegration data of D107V, D164H, E200D, and E200H are not shown.