Identification of Critical Motifs within HIV-1 Integrase Required for Importin alpha 3 Interaction and Viral cDNA Nuclear Import

• Published in: Journal of molecular biology 2011-07, Vol.410 (5), p.847-862
• Main Authors: Jayappa, Kallesh Danappa, Ao, Zhujun, Yang, Ming, Wang, Junzhi, Yao, Xiaojian
• Format: Article
• Language: English
• Subjects: Human immunodeficiency virus 1
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2011.04.011

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 INKK215,9AA (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 super(211KELQKQITK and ) super(2)62RRKAK regions of IN C-terminal domain are required for Imp alpha 3 interaction and HIV-1 cDNA nuclear import.