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CRISPR/Cas9-Induced Mutagenesis Corroborates the Role of Transportin-SR2 in HIV-1 Nuclear Import.
Janssens, Julie; Blokken, Jolien; Lampi, Yulia; De Wit, Flore; Zurnic Bonisch, Irena; Nombela, Ivan; Van de Velde, Paulien; Van Remoortel, Barbara; Gijsbers, Rik; Christ, Frauke; Debyser, Zeger.
Afiliação
  • Janssens J; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Blokken J; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Lampi Y; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • De Wit F; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Zurnic Bonisch I; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Nombela I; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Van de Velde P; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Van Remoortel B; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Gijsbers R; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Christ F; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
  • Debyser Z; Laboratory of Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Flanders, Belgium.
Microbiol Spectr ; 9(2): e0133621, 2021 10 31.
Article em En | MEDLINE | ID: mdl-34612665
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 ΔV105 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.
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Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Núcleo Celular / HIV-1 / Beta Carioferinas Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Microbiol Spectr Ano de publicação: 2021 Tipo de documento: Article País de afiliação: Bélgica

Texto completo: 1 Bases de dados: MEDLINE Assunto principal: Núcleo Celular / HIV-1 / Beta Carioferinas Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Microbiol Spectr Ano de publicação: 2021 Tipo de documento: Article País de afiliação: Bélgica