The incoming influenza A virus (IAV) genome must pass through two distinct barriers in order to establish infection in the cell: the plasma membrane and the nuclear membrane. A precise understanding of the challenges imposed by the nuclear barrier remains outstanding. Passage across is mediated by host karyopherins (KPNAs), which bind to the viral nucleoprotein (NP) via its N-terminal nuclear localization sequence (NLS). The binding affinity between the two molecules is low, but NP is present in a high copy number, which suggests that binding avidity plays a compensatory role during import. Using nanobody-based technology, we demonstrate that a high binding avidity is required for infection, though the absolute value differs between cell types and correlates with their relative susceptibility to infection. In addition, we demonstrate that increasing the affinity level caused a decrease in avidity requirements for some cell types but blocked infection in others. Finally, we show that genomes that become frustrated by low avidity and remain cytoplasmic trigger the type I interferon response. Based on these results, we conclude that IAV balances affinity and avidity considerations in order to overcome the nuclear barrier across a broad range of cell types. Furthermore, these results provide evidence to support the long-standing hypothesis that IAV's strategy of import and replication in the nucleus facilitates immune evasion.IMPORTANCE We used intracellular nanobodies to block influenza virus infection at the step prior to nuclear import of its ribonucleoproteins. By doing so, we were able to answer an important but outstanding question that could not be addressed with conventional tools: how many of the ∼500 available NLS motifs are needed to establish infection? Furthermore, by controlling the subcellular localization of the incoming viral ribonucleoproteins and measuring the cell's antiviral response, we were able to provide direct evidence for the long-standing hypothesis that influenza virus exploits nuclear localization to delay activation of the innate immune response.
Keywords: KPNA; NLS; NP; VHH; influenza A virus; interferon; nanobody; nuclear import.
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