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In vivo analysis of importin alpha proteins reveals cellular proliferation inhibition and substrate specificity.

ABSTRACT: The "classical" nuclear import pathway depends on importins alpha and beta. Humans have only one importin beta, while six alpha importins have been described. Whether or not distinct alpha importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha importins in HeLa cells. Down-regulation of importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha importin, indicating that the importin alpha/beta pathway was generally not affected. In contrast, importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin alpha3 in both cases. Thus, importin alpha3 is the only alpha importin responsible for the classical nuclear import of RCC1 in living cells.

SUBMITTER: Quensel C

PROVIDER: S-EPMC529023 | biostudies-other | 2004 Dec

REPOSITORIES: biostudies-other

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Project description:Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -β1 (Imp-β1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-β1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/β1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/β1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.

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In vivo analysis of importin alpha proteins reveals cellular proliferation inhibition and substrate specificity.

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