Collaborative studies reveal the replication structure of influenza viruses, revealing new discoveries and potential drug targets

A team of scientists at the University of Oxford used multiple techniques from the UK's National Synchrotron Diamond Light Source to address the structure of the flu's replication mechanism and determine how it interacts with cellular proteins
.
The new study further deepens the understanding
of influenza replication and how the virus adapts to different hosts.
These structural insights reveal new potential drug targets that can be used to develop novel antiviral drugs
that inhibit influenza virus replication.

The Diamond Electron Bioimaging Center (eBIC) published studies
using X-ray crystallography and small-angle X-ray scattering (ssaxophone) in synchrotrons and cryo-electron microscopy (cryo-em).
The paper, which will be published in the March 2023 issue, features an image
of a machine that replicates the flu virus on the cover.

In addition to causing seasonal flu, it can become a pandemic
when the flu jumps from animals to humans.
By looking more closely at the virus's replication cycle, researchers are piecing together how influenza viruses hijack human and animal cells for replication
.
This research is critical
to understanding how cellular proteins (ANP32A) explain host jumping disorders to some extent.
By studying which regions of viral polymerase interact with ANP32A, the researchers determined that mutations in avian influenza polymerase may allow it to interact with human ANP32A, allowing avian influenza strains to jump into the human ^host2.

Influenza viruses store their genes in RNA and synthesize their own RNA polymerase to replicate their genome
.
In addition to replication, the viral polymerase has a variety of functions, which Diamond's collaborative research helped elucinate.

These studies show that polymerases regulate transcription (the first step in protein synthesis) and the timing of replication, which can only begin
after viral proteins are produced.
The results reveal how polymerase interacts with the cellular protein ANP32A and uses it to protect viral RNA from detection
by the immune system.

The currently circulating influenza A virus is thought to be an evolutionary descendant of the virus that caused the 1918-1919 global influenza pandemic, which killed between 50 million and 100 million people
worldwide.
Influenza viruses are usually limited to infecting one animal host, such as birds, and require specific adaptations to jump to different animals, such as humans
.
The 1918 influenza virus, thought to have been transmitted from waterfowl to humans, is considered the "founder virus," contributing fragments
of the viral genome to all subsequent epidemics and pandemic strains.
In a study published earlier this year, the team determined the structure of the polymerase of the 1918 pandemic influenza virus and identified sites on the polymerase surface that were sensitive to inhibition1.
This, in turn, helps identify and validate targets for drug discovery
.

This research is crucial
for understanding how ANP32A partially explains host jumping disorders.
ANP32A is very different between humans and birds, forcing animals and avian influenza viruses to evolve less similarly
.
Structural biology research at Diamond University provides insight
into the pandemic potential of different influenza strains.
By studying which regions of viral polymerase interact with ANP32A, the researchers determined that mutations in avian influenza polymerase may allow it to interact with human ANP32A, allowing avian influenza strains to jump into the human ^host2.

The structural characterization of large protein complexes is a challenge, and influenza replication complexes are no exception
.
The near-atomic detail structure of viral polymerase was determined by X-ray crystallography on beamlines I03 and I24, revealing that individual polymerases paired to form dimers
.
To complement the crystal structure of the dimers, a structural technique called SAXS is performed in solution at the B21 beam to demonstrate the importance of
dimer formation for polymerase function.

The researchers propose that individual RNA polymerases are transcribed early in infection and switch to replication only when they are bound together as dimers, after producing additional copies of the ^polymerase3.

To further extend this structural work, the research team conducted low-temperature electromagnetic tests
at eBIC.
Professor Jonathan Grimes of the University of Oxford explains: "Cryo-EM allowed us to start looking at very interesting protein complexes, and we found that it was impossible to grow crystals in the laboratory"
.

Cryo-electron microscopy was used to determine the interaction between RNA and viral polymerase, revealing that one polymerase in the dimer replicates the viral genome, while another polymerase envelops the newly formed RNA in a viral protein, shielding it from immunosensors
.
Interestingly, influenza viruses hijacked the cellular protein ANP32A to stabilize dimers and assist in encapsulating and hiding viral RNA to avoid immunodetection
.

"The science of democratizing diamonds," Grimes explains
.
"All of these technologies exist in one place and are available to the scientific community, which is an invaluable resource
.
" These world-class, cutting-edge facilities are available free of charge to scientists from universities and institutes in the UK and the EU who have interesting and important biological questions"
.

Corresponding author Professor Ervin Fodor of the University of Microbiology of the University of Oxford concludes: "These studies help us identify and validate targets for drug discovery
.
We hope that the new understanding of influenza virus transcription mechanisms using Diamond's technology will eventually lead to novel antiviral drugs against influenza polymerase.
"