The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets.

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Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis.

Influenza A viruses cause pandemics when they cross between species and an antigenically novel virus acquires the ability to infect and transmit between these new hosts. The timing of pandemics is currently unpredictable but depends on ecological and virological factors. The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. These include the ability to bind and enter cells, to replicate the viral RNA genome within the host cell nucleus, to evade host restriction factors and innate immune responses and to transmit between individuals. In this Review, we examine the host barriers that influenza A viruses of animals, especially birds, must overcome to initiate a pandemic in humans and describe how, on crossing the species barrier, the virus mutates to establish new interactions with the human host. This knowledge is used to inform risk assessments for future pandemics and to identify virus-host interactions that could be targeted by novel intervention strategies.

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Host and viral determinants of influenza A virus species specificity.

Baloxavir acid (BXA), derived from the prodrug baloxavir marboxil (BXM), potently and selectively inhibits the cap-dependent endonuclease within the polymerase PA subunit of influenza A and B viruses. In clinical trials, single doses of BXM profoundly decrease viral titers as well as alleviating influenza symptoms. Here, we characterize the impact on BXA susceptibility and replicative capacity of variant viruses detected in the post-treatment monitoring of the clinical studies. We find that the PA I38T substitution is a major pathway for reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC50 changes in A and B viruses, respectively. The viruses harboring the I38T substitution show severely impaired replicative fitness in cells, and correspondingly reduced endonuclease activity in vitro. Co-crystal structures of wild-type and I38T influenza A and B endonucleases bound to BXA show that the mutation reduces van der Waals contacts with the inhibitor. A reduced affinity to the I38T mutant is supported by the lower stability of the BXA-bound endonuclease. These mechanistic insights provide markers for future surveillance of treated populations.

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Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil.

■ CONTENTS Strategies To Overcome Inherent Challenges in XLMS Studies C Bioinformatics Tools for Cross-Linked Peptide Analysis C Identification of Cross-Linked Peptides C Automated Visualization of Cross-Linked Residues D Development of MS-Cleavable Cross-Linking Reagents D Multistage Mass Spectrometry-Based XL-MS Analysis Workflow D MS-Based XL-MS Workflow F Other MS-Cleavable Cross-Linkers F Enrichment of Cross-Linked Peptides To Enhance Their Detection F Chromatographic Separation of Cross-Linked Peptides G Affinity Purification of Cross-Linked Peptides G Developing New Cross-Linking Chemistries G Acidic Residue-Specific Cross-Linkers G Nonspecific, Photoactivated Cross-Linkers H XL-MS Strategies for Structural Analysis of Protein Complexes H Combinatory Cross-Linking Strategies To Obtain Comprehensive Information H Sample Preparation for in Vitro XL-MS Analysis of Protein Complexes I In Vitro On-Bead Cross-Linking Strategies I Two-Step Cross-Linking Protocols J In Vivo Cross-Linking of Protein Complexes J Elucidation of Protein Complex Architectures J Coupling XL-MS with Other MS-Based Structural Tools K Integrative Modeling of Macromolecular Machines K 26S Proteasome L Nuclear Pore Complex M RNA Polymerase Complexes M Proteome-Wide XL-MS Studies M In Vitro Proteome-Wide Studies M In Vivo Proteome-Wide Studies O Quantitative XL-MS Strategies and Their Applications O Cross-Linking Reagent Labeling O Metabolic Labeling O Isobaric Labeling for Multiplexed Quantitation P Future Perspectives and Conclusions P Author Information Q Corresponding Author Q ORCID Q Notes Q Biographies Q Acknowledgments Q References Q

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Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology.

Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry-a review.

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of Pb2 Domains.

https://rd.springer.com/content/pdf/10.1016%2Fj.jasms.2009.09.005.pdf

Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions

Epstein-Barr virus (EBV) is a ubiquitous human pathogen which establishes life-long persistent infection in the large majority of the human population. During viral latency, the cellular machinery takes care of the replication of the viral episome. But EBV, as well as herpesviruses in general, codes for numerous enzymes required for lytic DNA replication which allow viral replication in resting cells. Recently, several tridimensional structures of these enzymes became available for EBV as well as for other herpesviruses so that structural information now exists for most of them. The replication process and the structures of the proteins involved in replication are reviewed in the light of potential drug development and of herpesvirus evolution. The structures of the proteins involved in lytic replication show the relationship between herpesviruses and tailed bacteriophages, furthermore they show that EBV proteins tend to be more complex than their counterparts in other organisms. In this review, we could show the phylogenetic position of the herpesvirus helicase close to the Dda helicases involved in initiation of replication of the caudovirales.

Eric Thierry

Papers

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of Pb2 Domains.

Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions

Structure des enzymes de réplication du virus Epstein-Barr