A team of scientists from the University of Oxford used multiple techniques at the Diamond Light Source, the UK’s national synchrotron, to reveal the structure of the influenza replication machinery and determine how it interacts with cellular proteins. This new research contributes to the understanding of influenza replication and how the virus adapts to different hosts. These structural insights revealed new potential drug targets for the development of novel antiviral drugs to inhibit influenza virus replication.
The paper “Structural understanding of influenza virus genome replication.” Trends in Microbiology (2022). DOI: 10.1016/j.tim.2022.09.015 outlines the findings generated using X-ray crystallography and small-angle X-ray scattering (SAXS) in synchrotron and cryo-electron microscopy (cryo-EM) at the Diamond Center for Electron Bioimaging ( eBIC). The paper will appear in print in the March 2023 issue with an image of the influenza virus replication machinery on its front cover.
In addition to causing seasonal flu, flu can become a pandemic when it jumps from animals to humans. By taking a closer look at the virus’ replication cycle, the researchers pieced together how influenza hijacks human and animal cells for its replication. This research is critical to understanding how a cellular protein (ANP32A) partially accounts for the host-jumping barrier. By examining which regions of the viral polymerase interact with ANP32A, the researchers found that a mutation in the avian flu polymerase could allow it to interact with human ANP32A, allowing this strain of avian flu to pass into human hosts2.
The influenza virus stores its genes in RNA, and the virus synthesizes its own RNA polymerase to replicate its genome. This viral polymerase has multiple functions in addition to replication, which collaborative research at Diamond has helped elucidate. These studies show that the polymerase regulates the timing of transcription – the first step in protein synthesis – and replication, which can only begin after viral proteins have been produced. The findings reveal how the polymerase interacts with a cellular protein, ANP32A, and tailors it to protect the viral RNA from detection by the immune system.
Influenza A viruses currently circulating are believed to be the evolutionary offspring of the virus that caused the global pandemic of 1918-1919, which was responsible for between 50 and 100 million deaths worldwide. Influenza viruses are usually limited to infecting one type of animal host, such as birds, and require specific adaptations to move to another animal, such as humans. The 1918 influenza virus is believed to have jumped from waterfowl to humans and is considered the “foundation virus” that contributed viral genome segments to all subsequent epidemic and pandemic strains. In a study published earlier this year, the group determined the structures of the polymerase from the 1918 pandemic influenza virus and identified sites on the surface of the polymerase that are sensitive to inhibition1. This, in turn, can help identify and validate drug discovery targets.
This study is critical to understanding how ANP32A partially accounts for the host hopping barrier. ANP32A differs significantly between humans and birds, forcing animal and avian influenza viruses to evolve to become less alike. Structural biology research at Diamond provides insight into the pandemic potential of different influenza strains. By examining which regions of the viral polymerase interact with ANP32A, the researchers found that a mutation in the avian flu polymerase could allow it to interact with human ANP32A, allowing this strain of avian flu to pass into human hosts2.
Structural characterization of large protein complexes is challenging, and the influenza replication complex was no exception. X-ray crystallography at the I03 and I24 beamlines was used to determine the structure of the viral polymerase in near-atomic detail, revealing that single polymerases pair to form dimers. To complement the crystal structure of the dimers, a structural technique known as SAXS was performed in solution on line B21 to demonstrate the importance of dimer formation for polymerase function.
The researchers proposed that single RNA polymerases carry out transcription early in infection and switch to replication later only when they bind together as dimers, after producing additional copies of the polymerase3.
To further extend this structural work, the research team performed cryo-EM at the eBIC. Professor Jonathan Grimes of the University of Oxford explains: “Cryo-EM has allowed us to start looking at very interesting protein complexes that we would have thought impossible to grow crystals in the lab.”
Interactions between RNA and viral polymerase were determined using cryo-EM, revealing that one polymerase in the dimer replicates the viral genome, while the other coats the nascent RNA in viral proteins that protect it from immune sensors. Interestingly, influenza hijacks the cellular protein ANP32A to stabilize the dimers and help coat and hide the viral RNA from immune detection.
“The diamond democratizes science,” Grimes explains. “The fact that all these techniques exist in one place and are available to the scientific community is an extremely valuable resource. These state-of-the-art, world-class facilities are freely available to scientists from UK and EU universities and institutes with interesting and important biological questions.”
Corresponding author of Trends in Microbiology review, Professor Erwin Fodor, University of Oxford, concluded: “These studies help us identify and validate drug discovery targets. It is hoped that the new insights generated into the workings of the influenza virus transcription machinery using the technologies at Diamond will eventually lead to new antiviral agents targeting the influenza polymerase.”
Reference: Zhu Z, Fodor E, Keown JR. Structural understanding of influenza virus genome replication. Trends Microbiol. 2022. doi: 10.1016/j.tim.2022.09.015
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