Structures of Nicotiana benthamiana importin-α1 complexes reveal mechanisms for plant virus NLS recognition.

Importin-α mediates nuclear protein import by recognizing nuclear localization signals (NLSs) and is frequently exploited by plant viruses to access the host nucleus. How plant importin-α maintains selectivity for endogenous cargos while accommodating diverse viral NLSs remains unclear. Here, we report the 1.88 Å crystal structure of Nicotiana benthamiana importin-α1 lacking the autoinhibitory IBB domain (NbImpα1ΔIBB), and its complexes with three plant viral NLSs: BBSV CP, CMV-fny 2b, and BBSV p7a, respectively. NbImpα1 adopts the canonical armadillo repeat-based superhelical fold with conserved major and minor NLS-binding sites. All viral NLS peptides bind in a 2:1 stoichiometry and share a common orientation along the central groove (C to N direction), but exhibit distinct interaction strategies. BBSV CP and CMV-fny 2b contain bipartite NLSs with reversed site usage, whereas BBSV p7a employs a monopartite NLS with repeated basic motifs to occupy both sites. Structural and functional analyses demonstrate that NbImpα1 decodes the three-dimensional spatial topology of basic residues rather than primary NLS sequences. The major binding site mediates high-specificity initial anchoring, while the minor binding site provides plasticity for diverse NLS recognition. This dual-site, topology-based model explains selective yet flexible nuclear import and viral adaptation to plant constraints.