Identification of Critical Motifs within HIV-1 Integrase Required for Importin α3 Interaction and Viral cDNA Nuclear Import

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin alpha 3 (Imp alpha 3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Imp alpha 3. Our results revealed a reduced interaction between the two IN mutants lNKK215,9AA(IN215,9) and INRK263,4AA (IN263,4) with Imp alpha 3, while an IN double mutant, IN215,9/263,4,was severely impaired for its Imp alpha 3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Imp alpha 3 are involved in interaction with IN. All of these results suggest a cargo protein import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that (211)KELQKQITK and (262)RRKAK regions of IN C-terminal domain are required for Imp alpha 3 interaction and HIV-1 cDNA nuclear import. (C) 2011 Elsevier Ltd. All rights reserved.