Influenza virus polymerase subunits co-evolve to ensure proper levels of dimerization of the heterotrimer


Autoři: Kuang-Yu Chen aff001;  Emmanuel Dos Santos Afonso aff001;  Vincent Enouf aff001;  Catherine Isel aff001;  Nadia Naffakh aff001
Působiště autorů: Unité de Génétique Moléculaire des Virus à ARN, Institut Pasteur, UMR 3569 CNRS, Paris, France aff001;  Unité de Génétique Moléculaire des Virus à ARN, Université Paris Diderot, Sorbonne Paris Cité, Paris, France aff002;  Unité de Génétique Moléculaire des Virus à ARN, Centre National de Référence des Virus des Infections Respiratoires, Institut Pasteur, Paris, France aff003;  Pasteur International Bioresources network (PIBnet), Plateforme de Microbiologie Mutualisée (P2M), Institut Pasteur, Paris, France aff004
Vyšlo v časopise: Influenza virus polymerase subunits co-evolve to ensure proper levels of dimerization of the heterotrimer. PLoS Pathog 15(10): e1008034. doi:10.1371/journal.ppat.1008034
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008034

Souhrn

The influenza A virus RNA-dependent RNA polymerase complex consists in three subunits, PB2, PB1 and PA, that perform transcription and replication of the viral genome through very distinct mechanisms. Biochemical and structural studies have revealed that the polymerase can adopt multiple conformations and form oligomers. However so far it remained unclear whether the available oligomeric crystal structures represent a functional state of the polymerase. Here we gained new insights into this question, by investigating the incompatibility between non-cognate subunits of influenza polymerase brought together through genetic reassortment. We observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone is attenuated, despite a 97% identity between the PR8-PB2 and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and/or PB2. The constellation of mutations present on one revertant virus was studied extensively using reverse genetics and cell-based reconstitution of the viral polymerase. The PA-E349K mutation appeared to play a major role in correcting the initial defect in replication (cRNA -> vRNA) of the PR8xWSN-PB2 reassortant. Strikingly the PA-E349K mutation, and also the PB2-G74R and PB1-K577G mutations present on other revertants, are located at a dimerization interface of the polymerase. All three restore wild-type-like polymerase activity in a minigenome assay while decreasing the level of polymerase dimerization. Overall, our data show that the polymerase subunits co-evolve to ensure not only optimal inter-subunit interactions within the heterotrimer, but also proper levels of dimerization of the heterotrimer which appears to be essential for efficient viral RNA replication. Our findings point to influenza polymerase dimerization as a feature that is controlled by a complex interplay of genetic determinants, can restrict genetic reassortment, and could become a target for antiviral drug development.

Klíčová slova:

Influenza A virus – Influenza viruses – Luciferase – Microbial mutation – Viral replication – Wireless sensor networks – Dimerization – Polymerases


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Hygiena a epidemiologie Infekční lékařství Laboratoř

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PLOS Pathogens


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