Molecular analysis of the contributions of human immunodeficiency virus type-1 integrase in post entry steps of early stage virus replication
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Date
2014-08-23
Authors
Danappa Jayappa, Kallesh
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Abstract
Human immunodeficiency virus type 1 (HIV-1) infection causes general loss of immune response in humans. Presently, an estimated 34 million (31.4-35.9 million) people worldwide are HIV-1 positive and many more are being newly infected. In the absence of a definitive cure, anti-HIV-1 drug therapy helps to manage the infection by suppressing virus replication. However, extensive drug resistance against most of existing drugs demands alternative anti-HIV-1 strategies. The proper knowledge about HIV-1 replication is essential to guide the development of new anti-HIV-1 strategies. The research presented in this thesis aims to understand the role of HIV-1 Integrase (IN) and cellular co-factors interactions in the early stage virus replication.
In the cytoplasm, HIV-1 cDNA exists as a high molecular weight nucleoprotein complex called pre-integration complex (PIC). The cDNA enters the nucleus as a part of PIC by active nuclear import and integrates into the host genome. HIV-1 Integrase (IN) protein has been recognized as a primary viral factor for HIV-1 nuclear import, but the key contributing cellular factor(s) is unknown. We have examined the requirement of different Importinα (Impα) isoforms for HIV-1 replication and identified the requirement of Impα3 for HIV-1 replication in HeLa cells, C8166T cells, and human macrophages. Further investigations showed the specific requirement of Impα3 for HIV-1 nuclear import. By analyzing the Impα3 interaction with HIV-1 proteins, we detected the IN interaction with Impα3 and C-terminal domain (CTD) of IN was essential for Impα3 interaction. These data led to the conclusion that Impα3 is required for HIV-1 nuclear import and interacts with IN. The IN-CTD consists of conserved basic amino acid rich motifs (211KELQKQITK, 236KGPAKLLWK, and 262RRKAK) that closely resemble the consensus classical nuclear localization signal (NLS) for Impα interaction. By substitution mutation and interaction analysis, 211KELQKQITK and 262RRKAK motifs in IN were identified as required for Impα3 interaction, IN nuclear localization, and HIV-1 nuclear import. Together, these data were useful in explaining the molecular mechanism of IN and Impα3 interaction and its requirement for HIV-1 nuclear import.
Retrograde transportation of macromolecules in the cytoplasm is one of the prerequisites for their nuclear import. Although an earlier study implicated the dynein complex in retrograde transport of HIV-1, cellular and viral factors that are involved in this process are unknown. In this study, we have elucidated the HIV-1 IN interaction with the dynein light chain 1 (DYNLL1) in 293T cells, in vitro, and in HIV-1 infected cells. DYNLL1 is one of the adapter proteins that mediate the cargo recruitment to dynein complex. However, our data suggested that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not for nuclear import. Surprisingly, DYNLL1 interaction of IN was dispensable for HIV-1 recruitment to dynein complex. These data led to the conclusion that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not required for HIV-1 recruitment to the dynein complex or for retrograde transport.
In summary, this study advances our knowledge on the role of IN and cellular factors interactions in different early steps of HIV-1 replication and offers potential contributions in the development of future anti-HIV-1 strategies.
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Keywords
HIV-1, Nuclear import, Uncoating, Reverse transcription, Integrase, Importin alpha3, Dynein light chain 1, Retrograde transportation