The influenza A virus RNA polymerase is a heterotrimer that transcribes and replicates the viral genome in the cell nucleus. Newly synthesized RNA polymerase subunits must therefore be imported into the nucleus during an infection. While various models have been proposed for this process, the consensus is that the polymerase basic protein PB1 and polymerase acidic protein PA subunits form a dimer in the cytoplasm and are transported into the nucleus by the beta-importin Ran-binding protein 5 (RanBP5), with the PB2 subunit imported separately to complete the trimeric complex. In this study, we characterized the interaction of PB1 with RanBP5 further and assessed its importance for viral growth. In particular, we found that the N-terminal region of PB1 mediates its binding to RanBP5 and that basic residues in a nuclear localization signal are required for RanBP5 binding. Mutating these basic residues to alanines does not prevent PB1 forming a dimer with PA, but does reduce RanBP5 binding. RanBP5-binding mutations reduce, though do not entirely prevent, the nuclear accumulation of PB1. Furthermore, mutations affecting RanBP5 binding are incompatible with or severely attenuate viral growth, providing further support for a key role for RanBP5 in the influenza A virus life cycle.

Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B’s therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.

Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1_MY) and E1191Q/S1199W (rBoNT/B1_QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1_MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.