Crystal structures of influenza nucleoprotein complexed with nucleic acid provide insights into the mechanism of RNA interaction

Abstract

The nucleoprotein (NP) of influenza virus is the core component of the ribonucleoprotein (RNP) and performs multiple structural and functional roles. Structures of the influenza A, B and D NP molecules have been solved previously, but structural information on how NP interacts with RNA remains elusive. Here we present the crystal structure of an obligate monomer of H5N1 NP in complex with RNA nucleotides to 2.3 Å, and a C-terminal truncation of this mutant, also in complex with RNA nucleotides, to 3 Å. In both structures, three nucleotides were identified near two positive grooves of NP suggested to be important for RNA binding. Structural evidence supports that conformational changes of flexible loops and the C-terminal tail both play important roles in the binding of RNA. Based on the structure, we propose a mechanism by which NP captures RNA by flexible loops and transfers it onto the positive binding grooves. Binding of RNA by NP is a crucial step for template re-encapsidation during transcription and replication and cRNP formation. Our structures thus provide insights into the molecular virology of the influenza virus.

The Role of Nucleoprotein in Immunity to Human Negative-Stranded RNA Viruses—Not Just Another Brick in the Viral Nucleocapsid

“…In fact, the NSV genomes are never free of NP in nature. For many NSVs, direct evidence on the RNA-binding mechanism has been proven by co-crystallization of the RNA–NP complexes [ 47 , 63 , 65 , 75 , 76 , 77 ]. Essential for the protective function of NP is its capacity to tightly bind the RNA and oligomerize and shape a helical RNC.…”

H7N9 bearing a mutation in the nucleoprotein leads to increased pathology in chickens

“…Furthermore, we mapped this change to a predicted structure of the NP protein and we were able to demonstrate that it is present on an exposed surface ( Figure 3D ). It has been demonstrated that the NP 360-373 loop is critical in RNA binding ( 21 ). Importantly, none of the viral isolates had incorporated multiple basic amino acids in the cleavage site of the HA protein ( Figure 3A ), which is recognized as the major determinant of AI virus pathogenicity.…”

“…Therefore, the changes we have observed in the NP may have led to increased pathology through changes in peptide/MHCI-TCR interactions. In addition to the impact on T cell subtypes, the mutation in the NP protein it has been demonstrated that the NP 360-373 loop is critical in RNA binding ( 21 ). This role in the stabilization of RNA is an essential role of NP in replication of the virus and may lead to increased proliferation potential.…”

Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

“…2 ). Because both NP mutants contain alanine substitutions in residues 213, 214, and 216, which are located close to the RNA-binding groove formed by R74, R75, R174, R175, and R221 (NP-G1 region) ( 31 – 33 ), such mutations potentially alter the RNA-binding property of NP and subsequent RNP formation ( 34 ). However, NoLS-NP NoLSmut , which contains alanine substitutions in the intrinsic NoLS but has an ectopic NoLS at the amino terminus, had the ability to form functional double-helical RNPs (see Fig.…”