Dataset Information

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

ABSTRACT: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans^1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol_A) composed of PB1, PB2 and PA subunits^3-5. Although the high-resolution crystal structure of FluPol_A of bat influenza A virus has previously been reported⁶, there are no complete structures available for human and avian FluPol_A. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase^7-10-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol_A from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol_A forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol_A bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol_A dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol_A dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol_A, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol_A that could be targeted in the development of antiviral drugs.

SUBMITTER: Fan H

PROVIDER: S-EPMC6795553 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Fan Haitian H Walker Alexander P AP Carrique Loïc L Keown Jeremy R JR Serna Martin Itziar I Karia Dimple D Sharps Jane J Hengrung Narin N Pardon Els E Steyaert Jan J Grimes Jonathan M JM Fodor Ervin E

Nature 20190904 7773

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3-5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been ...[more]

PMID: 31485076

Similar Datasets

Project description:The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular.IMPORTANCE Influenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterize the role of the flexible surface-exposed PA51-72-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.

Dataset Information

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Publications

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets