1. CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.
- Author
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Janssens J, Blokken J, Lampi Y, De Wit F, Zurnic Bonisch I, Nombela I, Van de Velde P, Van Remoortel B, Gijsbers R, Christ F, and Debyser Z
- Subjects
- Active Transport, Cell Nucleus, CRISPR-Cas Systems, Cell Nucleus metabolism, HIV Infections genetics, HIV Infections virology, HIV Integrase genetics, HIV Integrase metabolism, HIV-1 genetics, Humans, Protein Binding, Serine-Arginine Splicing Factors genetics, Serine-Arginine Splicing Factors metabolism, beta Karyopherins genetics, mRNA Cleavage and Polyadenylation Factors genetics, mRNA Cleavage and Polyadenylation Factors metabolism, Cell Nucleus virology, HIV-1 metabolism, beta Karyopherins metabolism
- Abstract
To infect nondividing cells, HIV-1 needs to cross the nuclear membrane. The importin transportin-SR2 (TRN-SR2 or transportin-3) has been proposed to mediate HIV-1 nuclear import, but the detailed mechanism remains unresolved. The direct interaction of TRN-SR2 with HIV-1 integrase (IN) has been proposed to drive HIV-1 nuclear import. Alternatively, TRN-SR2 may play an indirect role by mediating nuclear import of cleavage and polyadenylation specificity factor 6 (CPSF6). To unravel the role of TRN-SR2, we designed CRISPR/Cas9 guide RNAs targeting different exons of TNPO3 . Although this approach failed to generate full knockouts, monoallelic knockout clones were generated with indel mutations. HIV-1 replication was hampered in those clones at the level of HIV-1 nuclear import without an effect on the cellular distribution of the TRN-SR2 cargoes CPSF6 or alternative splicing factor1/pre-mRNA splicing factor SF2 (ASF/SF2). Recombinant ΔV
105 TRN-SR2 expressed in clone 15.15 was 2-fold impaired for interaction with HIV-1 IN and classified as an interaction mutant. Our data support a model whereby TRN-SR2 acts as a cofactor of HIV-1 nuclear import without compromising the nuclear import of cellular cargoes. CRISPR/Cas9-induced mutagenesis can be used as a method to generate interface mutants to characterize host factors of human pathogens. IMPORTANCE Combination antiretroviral therapy (cART) effectively controls HIV-1 by reducing viral loads, but it does not cure the infection. Lifelong treatment with cART is a prerequisite for sustained viral suppression. The rapid emergence of drug-resistant viral strains drives the necessity to discover new therapeutic targets. The nuclear import of HIV-1 is crucial in the HIV-1 replication cycle, but the detailed mechanism remains incompletely understood. This study provides evidence that TRN-SR2 directly mediates HIV-1 nuclear import via the interaction with HIV-1 integrase. The interaction between those proteins is therefore a promising target toward a rational drug design which could lead to new therapeutic strategies due to the bottleneck nature of HIV-1 nuclear import.- Published
- 2021
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