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The role of the priming loop in influenza A virus RNA synthesis

Nature Microbiology volume 1, Article number: 16029 (2016) Cite this article

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Abstract

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1. They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb1,2. Conserved RdRp motifs A–F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation1. For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.

RNA virus RNA-dependent RNA polymerases (RdRps) catalyse the formation of the first phosphodiester bond of the nascent strand either de novo or in a primer-dependent manner1. A key structural feature that differentiates these two mechanisms is the priming loop, which is a loop structure that protrudes from the palm5 or thumb subdomain6,7 towards the active site of de novo-initiating RdRps. Strictly primer-dependent RdRps lack such a structure8. An important function of the priming loop is to act as stacking platform for the initiating NTP during de novo initiation on the 3′ terminus of the bound single-stranded RNA (ssRNA) template6,9, i.e. to ensure terminal initiation. The interaction between the NTP and loop is typically mediated by a conserved aromatic residue, which buttresses the sugar–base of the initiating NTP6,9,10. A second role of the priming loop is to provide selection against double-stranded RNA (dsRNA) and ensure that only an ssRNA terminus is positioned into the active site10–12. This is consistent with the observation that the priming loop undergoes a conformational change, either retracting like a compressed spring9 or folding into the minor groove of the product duplex5, to allow egress of a product duplex.

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Figure 1: The PB1 β-hairpin is essential for influenza A virus RNA synthesis in cell culture and terminal de novo replication initiation in vitro
Figure 2: The PB1 β-hairpin loop is not essential for extension during replication.
Figure 3: The PB1 β-hairpin is not essential for transcription initiation.
Figure 4: PB1 P651 is essential for terminal de novo initiation during replication.

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Acknowledgements

This work was supported by a Wellcome Trust grant 098721/Z/12/Z (to A.J.W.t.V.), a Netherlands Organization for Scientific Research (NWO) grant 825.11.029 (to A.J.W.t.V.), a Medical Research Council (MRC) grant MR/K000241/1 (to E.F.), a European Commission Seventh Framework Program grant FP7/2007-2013 HEALTH-F4-2008-201418 (to A.N.K.), a Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/J001694/1 (to A.N.K. and E.F.), and a Kemp postdoctoral fellowship from Lincoln College Oxford (to A.J.W.t.V.).

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Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

    Aartjan J. W. te Velthuis & Ervin Fodor

  2. Department of Physics, Clarendon Laboratory, University of Oxford, Parks Road, Oxford OX1 3PU, UK

    Aartjan J. W. te Velthuis, Nicole C. Robb & Achillefs N. Kapanidis

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  1. Aartjan J. W. te Velthuis
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Contributions

A.J.W.t.V. and N.C.R. designed experiments. A.J.W.t.V. performed experiments. A.J.W.t.V., N.C.R., A.N.K. and E.F. analysed data. A.J.W.t.V. and E.F. wrote the manuscript.

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Correspondence to Aartjan J. W. te Velthuis.

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Supplementary Figures 1-18, Tables 1-3 and References (PDF 20544 kb)

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te Velthuis, A., Robb, N., Kapanidis, A. et al. The role of the priming loop in influenza A virus RNA synthesis. Nat Microbiol 1, 16029 (2016). https://doi.org/10.1038/nmicrobiol.2016.29

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