Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation

Abstract

Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo–electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein

“…Together, these residues form a positively charged patch located between the NP head and body domain ( Fig. S1B ), which is thought to interact with the negatively charged viral RNA backbone ( 14 , 16 , 19 , 20 ). It was previously shown that during viral infection, K184 and K229 undergo post-translational modifications that remove the positive charge from their side chain, namely, acetylation (for K184 and K229) and ubiquitination (for K184) ( 22 , 24 ).…”

“…NP serves as the structural scaffold of vRNPs and consists of a head domain, a body domain, and a tail loop ( 14 ). Within vRNPs, each NP protomer binds approximately 12 nucleotides ( 15 , 16 ), and vRNA regions located between such NP-bound sites tend to form secondary structures. NP binds the vRNAs unevenly and without clear sequence or structure specificity ( 17 , 18 ), possibly through electrostatic interactions between a positively charged RNA-binding groove located between the head and body domains of NP and the negatively charged sugar-phosphate backbone of the vRNAs ( 14 , 19 , 20 ).…”

Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments

“…However, NP crystal structures revealed a putative RNA binding groove between the head and body domains of NP lined with conserved basic residues that most likely interact with the ribose phosphate moieties of RNA [ 12 , 15 , 16 , 17 ]. Furthermore, a recent sub-nanometric structure of a model helical IAV nucleocapsid identified RNA densities adjacent and between the positively charged NP surfaces [ 20 ]. Therefore, since NMIA, as all SHAPE reagents, modifies the 2′OH of unpaired and flexible nucleotides, it is expected that the binding of NP decreases the SHAPE reactivity of the nts interacting with the protein.…”

“…The vRNA structure is not visible in cryo-EM structures of authentic vRNPs [ 3 , 51 , 52 ], and while recent structures of NP/RNA complexes [ 20 , 59 ] help us understand how vRNPs are organized, they do not reveal how RNA structures can protrude from the central vRNP helical structure. Therefore, chemical probing [ 45 , 46 ] remains an important tool for the investigation of the impact of NP on the vRNA structure.…”

“…NP binds single-stranded RNA (ssRNA) with high affinity and little or no sequence specificity [ 14 , 18 ]. Each NP protomer binds ~12 nts [ 19 ], and IAV NP complexed with a 12-mer RNA assembles into long filaments resembling authentic vRNPs [ 14 , 20 ].…”