Crystal structure of the RNA-dependent RNA polymerase from influenza C virus

Hengrung, Narin; Omari, Kamel El; Martin, I. Serna; Vreede, Frank T.; Cusack, Stephen; Rambo, Robert P.; Vonrhein, Clemens; Bricogne, G.; Stuart, David I.; Grimes, Jonathan M.; Fodor, Ervin

doi:10.1038/nature15525

Cited by 154 publications

(191 citation statements)

References 44 publications

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“…This is consistent with the location of the 627 domain on the outside of the polymerase core, which is made up of PB1, the C-terminal domain of PA, and an N-terminal third of PB2 (11–13). The 627 domain is also not required for RNA template binding, as the RNA-binding functions reside in the polymerase core (12, 13).…”

Section: Discussionsupporting

confidence: 84%

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

Nilsson

Velthuis

Fodor

2017

J Virol

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The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

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Section: Discussionsupporting

confidence: 84%

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

Nilsson

Velthuis

Fodor

2017

J Virol

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“…Nonetheless, the function of this loop region is not completely clear. It is suspected to interact with the viral genome in the resolved influenza B and C virus structures (46, 47, 84), and K198 of influenza A virus was suggested to be related to host adaptation (85). As the density of the loop region (residues 195 to 198) is missing from the influenza A virus polymerase crystal structure, we used kinematic loop modeling in the Rosetta software to computationally reconstruct the loop region (86).…”

Section: Resultsmentioning

confidence: 99%

“…The PB2 structure is shaded in green, and the possible interacting residues of PB1 are blue. The interface residues were predicted by the SASA changing upon complex formation with Sppider (84). Download …”

Section: Supplemental Materialsmentioning

confidence: 99%

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

Jiang

et al. 2016

mBio

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Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

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“…Adaptive mutations increase replication, pathogenicity, and transmission of avian-origin viruses in mammalian hosts. Structural analyses have revealed that portions of PB2, including the 627 domain, remain solvent exposed in the holoenzyme and undergo large-scale conformational reorganization depending on whether the polymerase is replicating or transcribing (Hengrung et al, 2015; Pflug et al, 2014; Reich et al, 2014; Thierry et al, 2016). These data raise the possibility that adaptive mutations in PB2 may be important for intra- or inter-molecular protein:protein interactions and conformational rearrangements.…”

Section: Introductionmentioning

confidence: 99%

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

2018

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SUMMARY Adaptation of viruses to their hosts can result in specialization and a restricted host range. Species specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently identified as a cellular co-factor affecting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation among birds differentially affects viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

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Crystal structure of the RNA-dependent RNA polymerase from influenza C virus

Cited by 154 publications

References 44 publications

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

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