title: Identification of critical motifs within HIV-1 integrase required for importin α3 interaction and viral cDNA nuclear import.
abstract: 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 α3 (Impα3) 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 Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-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 Impα3 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 Impα3 interaction and HIV-1 cDNA nuclear import.
authors.affiliation: Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, 508-745 William Avenue, Winnipeg, Canada R3E 0J9.
authors.forename: Kallesh Danappa
authors.initials: KD
authors.lastname: Jayappa
authors.forename: Zhujun
authors.initials: Z
authors.lastname: Ao
authors.forename: Ming
authors.initials: M
authors.lastname: Yang
authors.forename: Junzhi
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Yao
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002455:Cell Division; D002467:Cell Nucleus / Q000821:virology*; D002522:Chlorocebus aethiops; D017124:Conserved Sequence / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D057809:HEK293 Cells; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology; D028901:alpha Karyopherins / Q000378:metabolism*
chemical_list: D024361:Amino Acids, Basic; D018076:DNA, Complementary; D004279:DNA, Viral; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D028901:alpha Karyopherins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1