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

Abstract

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

Keywords: NP; RNA chaperon; RNA structure; chemical probing; influenza A virus; nucleoprotein; vRNA.

Figure 1

Characterization of the WSN NP protein. Fractions obtained after the last chromatographic step performed at high [40] (a) or low ionic strength (b) were analyzed using SDS PAGE. The DLS profiles of the proteins purified in (a,b) are shown in panels (c,d), respectively. (e) Ten pmoles of NS (left) or M (right) vRNAs were incubated with increasing amounts of NP protein purified at low ionic strength at the nt/NP ratios indicated below the lanes and analyzed by electrophoresis through a 1% agarose gel containing 0.5 µg ethidium bromide per ml.

Figure 2

Comparison of the predicted secondary structures under the NoNP and ProtK conditions for the NS (a) and M (b) vRNAs. Structural elements that are common to the NoNP and ProtK conditions are indicated in grey, and those unique to the NoNP and ProtK conditions are in yellow and purple, respectively. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.

Figure 3

SHAPE reactivity differences under the ProtK versus NoNP condition. (a) NS vRNA; (b) M vRNA. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.

Figure 3

SHAPE reactivity differences under the ProtK versus NoNP condition. (a) NS vRNA; (b) M vRNA. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.

Figure 4

SHAPE reactivity differences under the Comp versus NoNP condition. (a) NS vRNA; (b) M vRNA. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.

Figure 4

SHAPE reactivity differences under the Comp versus NoNP condition. (a) NS vRNA; (b) M vRNA. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.

Figure 5

Base composition of the protected regions. (a) Base composition of all the protected regions (orange), the short protected regions (grey), and the long protected regions (yellow) compared to the composition of the NS and M vRNAs (dark blue). (b) Length distribution of the protected regions. Statistical tests of conformity were used to test whether the nt distribution in the protected regions differs from the nt distribution of the sequences of the NS and M vRNAs (a) or whether the observed distribution of the length of the protected regions differs from a random distribution of the protected nts along the vRNA sequences (b). *: p < 0.05.

Figure 6

Frequency of the 16 dinucleotides in the NS and M vRNA sequences and in the regions of the vRNAs that are protected upon NP addition. Dinucleotides are written from 3′ to 5′ as per convention in the field of negative strand viruses. A statistical test of conformity was used to compare the distribution of the 16 dinucleotides in the protected regions and in the NS and M vRNA sequences. Since the number of each dinucleotide in the protected region was low, p values < 0.10 (*) were considered significant.

Figure 7

Comparison of the predicted secondary structures of the NS (a) and M (b) vRNAs complexed with NP. SHAPE data were treated as hard constraints, partial pseudo-energies, or pseudo-energies (See main text for detailed explanation). Structural elements that are common to three or two predicted structures or exist solely in one structure are color-coded as indicated at the top of the panels. Nucleotides of the vRNA are numbered from 3′ to 5′ as per convention in the field of negative strand viruses.