Structural and Functional Motifs in Influenza Virus RNAs

Abstract

Influenza A viruses (IAV) are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the Orthomyxoviridae family and their genome consists of eight (-) sense viral RNA (vRNA) segments of different lengths coding for at least 11 viral proteins. A heterotrimeric polymerase complex is bound to the promoter consisting of the 13 5'-terminal and 12 3'-terminal nucleotides of each vRNA, while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP), thus forming ribonucleoproteins (vRNP). Transcription and replication of vRNAs result in viral mRNAs (vmRNAs) and complementary RNAs (cRNAs), respectively. Complementary RNAs are the exact positive copies of vRNAs; they also form ribonucleoproteins (cRNPs) and are intermediate templates in the vRNA amplification process. On the contrary, vmRNAs have a 5' cap snatched from cellular mRNAs and a 3' polyA tail, both gained by the viral polymerase complex. Hence, unlike vRNAs and cRNAs, vmRNAs do not have a terminal promoter able to recruit the viral polymerase. Furthermore, synthesis of at least two viral proteins requires vmRNA splicing. Except for extensive analysis of the viral promoter structure and function and a few, mostly bioinformatics, studies addressing the vRNA and vmRNA structure, structural studies of the influenza A vRNAs, cRNAs, and vmRNAs are still in their infancy. The recent crystal structures of the influenza polymerase heterotrimeric complex drastically improved our understanding of the replication and transcription processes. The vRNA structure has been mainly studied in vitro using RNA probing, but its structure has been very recently studied within native vRNPs using crosslinking and RNA probing coupled to next generation RNA sequencing. Concerning vmRNAs, most studies focused on the segment M and NS splice sites and several structures initially predicted by bioinformatics analysis have now been validated experimentally and their role in the viral life cycle demonstrated. This review aims to compile the structural motifs found in the different RNA classes (vRNA, cRNA, and vmRNA) of influenza viruses and their function in the viral replication cycle.

Keywords: CRNA; RNA; RNA structure; influenza; influenza A virus; promoter; vRNA.

FIGURE 1

(A) Schematic representation of the eight RNA segments of Influenza A viruses. AUG start codons of the different proteins are represented as green dots, while stop codons are indicated by red dots. Regions between U12/U13 and start codons correspond to the untranslated regions (UTRs). Below the M and NS segments are represented the spliced mRNAs. Important nucleotides positions are indicated. (B) Different vRNA promoter structure models proposed over time. Unpaired nucleotides are highlighted in yellow. In the ‘hook structure’ (4,6), two canonical base-pairs are flanked by two non-canonical A-A base-pairs.

FIGURE 2

X-ray structure of the vRNA promoter bound to the polymerase complex of bat IAV. The surface of the PA, PB1, and PB2 subunits are represented in magenta, violet-blue, and orange, respectively; the 5′ and 3′ strand of the vRNA promoter are drawn in cyan and yellow, respectively. This figure was drawn with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) using the coordinates obtained by Pflug et al. (2014) (Protein Data Bank accession number 4WSB). (A) General view of the complex. (B) and (C) Close up views of the promoter and the stem loop formed at the 5′ end of the 5′ strand. The 5′ end of the 5′ strand and the 3′ end of the 3′ strand of the promoter are labeled.

FIGURE 3

RNA structures identified along the segment M in both (–) and (+) strands. Their locations are indicated by dashed squares on the segment scheme at the top of the figure. (A) Similar stem-loop SL3 found in both strands at the exact same positions. (B) Other stem-loops found in this segment by bioinformatics analysis.

FIGURE 4

Kissing-loop interaction from between segments PB1 and NS of an avian H5N2 virus. These regions were identified to interact with each other by electrophoretic mobility shift assay and the folding of these structures suggests an interaction via a kissing-loop mechanism.

FIGURE 5

Alternative secondary structures at the 3′ splice site of the segment NS mRNA. Their locations are indicated by a dashed square on the segment scheme at the top of the figure. The red arrow indicates the 3′ splice site.

FIGURE 6

Different secondary structures proposed for the 5′ splice site of segment NS mRNA. Their locations are indicated by dashed squares on the segment scheme at the top of the figure. (A) The multi-branch structure proposed by Ilyinskii et al. (2009). (B) The tetraloop hairpin model presented by Moss et al. (2011). (C) The hairpin structure proposed and validated by chemical probing experiments by Priore et al. (2013a).

FIGURE 7

Alternative secondary structures at the 3′ splice site of the segment M mRNA. Their locations are indicated by a dashed square on the segment scheme at the top of the figure. The red arrowhead indicates the splice site.

FIGURE 8

Different secondary structures proposed for the 5′ splice site of segment 7 mRNA. Their locations are indicated by a dashed square on the segment scheme at the top of the figure. (A) Multi-branch structure proposed by Moss et al. (2011). (B) Double hairpin structure proposed by Jiang et al. (2014).

FIGURE 9

RNA secondary structure in the mRNA of segment 5. Predicted (A) and experimental (B) structures are almost identical. The red box shows the only difference, located in a loop, between these two structures. The region of interest is indicated by dashed square on the segment scheme at the top of the figure.