Solution structure of the influenza A virus cRNA promoter: implications for differential recognition of viral promoter structures by RNA-dependent RNA polymerase

Abstract

Influenza A virus replication requires the interaction of viral RNA-dependent RNA polymerase (RdRp) with promoters in both the RNA genome (vRNA) and the full-length complementary RNA (cRNA) which serve as templates for the generation of new vRNAs. Although RdRp binds both promoters effectively, it must also discriminate between them because they serve different functional roles in the viral life cycle. Even though the inherent asymmetry between two RNA promoters is considered as a cause of the differential recognition by the RdRp, the structural basis for the ability of the RdRp to recognize the RNA promoters and discriminate effectively between them remains unsolved. Here we report the structure of the cRNA promoter of influenza A virus as determined by heteronuclear magnetic resonance spectroscopy. The terminal region is extremely unstable and does not have a rigid structure. The major groove of the internal loop is widened by the displacement of a novel A*(UU) motif toward the minor groove. These internal loop residues show distinguishable dynamic characters, with differing motional timescales for each residue. Comparison of the cRNA promoter structure with that of the vRNA promoter reveals common structural and dynamic elements in the internal loop, but also differences that provide insight into how the viral RdRp differentially recognizes the cRNA and vRNA promoters.

Figure 1

(A) Strategies for mRNA synthesis and genomic replication of the viral RNA genome of influenza A virus. The newly synthesized cRNA associates with NP and RdRp to form a cRNP complex, which then produces vRNAs. The newly synthesized vRNAs then bind NP and RdRp to form a vRNP complex, which gives rise to viral mRNA. The mRNA strands have the canonical 5′ cap and 3′ poly(A) tail to allow nuclear export and translation by the host translation machinery. In contrast, the cRNAs do not have such modifications, but are full-length copies of the vRNA molecules. (B) Terminal sequences of vRNA from the influenza A virus. Boxed sequences are converged in all of the influenza A virus variants. Numbering of the 3′ strand is followed by a prime notation (′). The sequence shown is that of vRNA segment 8 of influenza A/PR/8/34. (C) Secondary structure of the 31 nt vRNA promoter, which was determined previously (left), and the 31 nt cRNA promoter (middle) and 25 nt cRNA promoter (right), which were studied in this paper. Watson–Crick base pairs and wobble base pairs are distinguished by bars and circles, respectively. The terminal 5 bp of the 31 nt cRNA promoter have been changed to two G-C pairs in the 25 nt cRNA promoter model; these are boxed.

Figure 2

(A) Superimposed imino region spectrum of the 31 nt cRNA promoter and 25 nt cRNA promoter. The spectrum was recorded at 4°C in 20 mM sodium phosphate buffer (pH 6.0) with 0.1 mM EDTA using a Bruker DRX 600 MHz spectrometer. (B) Fingerprint (aromatic-H1′) region in a NOESY spectrum of the 25 nt cRNA promoter; sequential assignments are indicated in violet. The spectrum was recorded at 300 K in 20 mM sodium phosphate buffer (pH 6.0) with 0.1 mM EDTA using a Bruker DRX 800 MHz spectrometer. The mixing time was 200 ms.

Figure 3

(A) Stereo view of the lowest energy member of the family of 31 converged structures. Adenines are colored in yellow, guanines in pink, uridines in green and cytidines in light blue. (B) Superimposed overall structures (from residue 5 to 21) of the 10 lowest energy members of the structure ensemble. (C) Superimposed internal loop structures of the 10 lowest energy members of the structure ensemble.

Figure 4

(A) Major groove stereoview of the internal loop of the cRNA promoter (top) and the vRNA promoter (PDB accession no. 1fo7). (B) Top views of the A*(UU) from the cRNA promoter (left) and (AA)*U from the vRNA promoter structure (right).

Figure 5

(A) Nucleotides within the influenza A virus cRNA promoter experience internal motion on different timescales. Normalized intensity of H6(8)/C6(8) cross-peaks versus ¹³C spin-lock mixing time in T1_ρ experiments for C6, C10, U12, U18, U19 and U22. The non-linear least squares fit of the data for each residue is shown with a solid line. (B) The plot of the number of NOE restraints per residue over the whole RNA. Sequential and long range NOE were counted both sides of two residues. A7, U18 and U19 are underlined. Total numbers of restraints of those residues are not significantly smaller than other residues, while the number of sequential NOEs of U19 is relatively small and most of the restraints of internal loop residues were given loosely.

Figure 6

The differential recognition of the vRNA and cRNA promoters by influenza RdRp. (A) The RdRp can bind to both the vRNA and cRNA promoters. (B) The endonuclease activity of the RdRp activated by the vRNA promoter requires the presence of (AA)*U in the internal loop and the stable terminal stem. (C) The packaging signal of vRNP is the (AA)*U in the internal loop of vRNA promoter.