New insights on how bird flu crosses the species barrier

In recent years, public health measures, surveillance, and vaccination have helped bring about significant progress in reducing the impact of seasonal flu epidemics, caused by human influenza viruses A and B. However, a possible outbreak of avian influenza A (commonly known as 'bird flu') in mammals, including humans, poses a significant threat to public health.

The Cusack group at EMBL Grenoble studies the replication process of influenza viruses. A new study from this group sheds light on the different mutations that the avian influenza virus can undergo to be able to replicate in mammalian cells.

Some avian influenza strains can cause severe disease and mortality. Fortunately, significant biological differences between birds and mammals normally prevent avian influenza from spreading from birds to other species. To infect mammals, the avian influenza virus must mutate to overcome two main barriers: the ability to enter the cell and to replicate within that cell. To cause an epidemic or pandemic, it must also acquire the ability to be transmitted between humans.

However, sporadic contamination of wild and domestic mammals by bird flu is becoming increasingly common. Of particular concern is the recent unexpected infection of dairy cows in the USA by an avian H5N1 strain, which risks becoming endemic in cattle. This might facilitate adaptation to humans, and indeed, a few cases of transmission to humans have been reported, so far resulting in only mild symptoms.

At the heart of this process is the polymerase, an enzyme that orchestrates the virus's replication inside host cells. This flexible protein can rearrange itself according to the different functions it performs during infection. These include transcription -- copying the viral RNA into messenger RNA to make viral proteins -- and replication -- making copies of the viral RNA to package into new viruses.

Viral replication is a complex process to study because it involves two viral polymerases and a host cell protein -- ANP32. Together, these three proteins form the replication complex, a molecular machine that carries out replication. ANP32 is known as a 'chaperone', meaning that it acts as a stabiliser for certain cellular proteins. It can do this thanks to a key structure -- its long acidic tail. In 2015, it was discovered that ANP32 is critical for influenza virus replication, but its function was not fully understood.

The results of the new study, published in the journal Nature Communications, show that ANP32 acts as a bridge between the two viral polymerases -- called replicase and encapsidase. The names reflect the two distinct conformations taken up by the polymerases to perform two different functions -- creating copies of the viral RNA (replicase) and packaging the copy inside a protective coating with ANP32's help (encapsidase).

Through its tail, ANP32 acts as a stabiliser for the replication complex, allowing it to form within the host cell. Interestingly, the ANP32 tail differs between birds and mammals, although the core of the protein remains very similar. This biological difference explains why the avian influenza virus does not replicate easily in mammals and humans.

"The key difference between avian and human ANP32 is a 33-amino-acid insertion in the avian tail, and the polymerase has to adapt to this difference," explained Benoît Arragain, a postdoctoral fellow in the Cusack group and first author of the publication. "For the avian-adapted polymerase to replicate in human cells, it must acquire certain mutations to be able to use human ANP32."

To better understand this process, Arragain and his collaborators obtained the structure of the replicase and encapsidase conformations of a human-adapted avian influenza polymerase (from strain H7N9) while they were interacting with human ANP32. This structure gives detailed information about which amino acids are important in forming the replication complex and which mutations could allow the avian influenza polymerase to adapt to mammalian cells.

To obtain these results, Arragain carried out in vitro experiments at EMBL Grenoble, using the Eukaryotic Expression Facility, the ISBG biophysical platform, and the cryo-electron microscopy platform available through the Partnership for Structural Biology. "We also collaborated with the Naffakh group at the Institut Pasteur, who carried out cellularexperiments," added Arragain. "In addition, we obtained the structure of the human type B influenza replication complex, which is similar to that of influenza A. The cellular experiments confirmed our structural data."

These new insights into the influenza replication complex can be used to study polymerase mutations in other similar strains of the avian influenza virus. It is therefore possible to use the structure obtained from the H7N9 strain and adapt it to other strains such as H5N1.

"The threat of a new pandemic caused by highly pathogenic, human-adapted avian influenza strains with a high mortality rate needs to be taken seriously," said Stephen Cusack, EMBL Grenoble Senior Scientist who led the study and has been studying influenza viruses for 30 years. "One of the key responses to this threat includes monitoring mutations in the virus in the field. Knowing this structure allows us to interpret these mutations and assess if a strain is on the path of adaptation to infect and transmit between mammals."

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Human cells help researchers understand squid camouflage

Squids and octopuses are masters of camouflage, blending into their environment to evade predators or surprise prey. Some aspects of how these cephalopods become reversibly transparent are still "unclear," largely because researchers can't culture cephalopod skin cells in the lab. Today, however, researchers report that they have replicated the tunable transparency of some squid skin cells in mammalian cells, which can be cultured. The work could not only shed light on basic squid biology, but also lead to better ways to image many cell types.

The researchers will present their results at the spring meeting of the American Chemical Society (ACS).

For many years, Alon Gorodetsky, Ph.D., and his research group have been working on materials inspired by squid. In past work, they developed "invisibility stickers," which consisted of bacterially produced squid reflectin proteins that were adhered onto sticky tape. "So then, we had this crazy idea to see whether we could capture some aspect of the ability of squid skin tissues to change transparency within human cell cultures," says Gorodetsky, who is the principal investigator on the project.

The team at the University of California, Irvine focused their efforts on cephalopod cells called leucophores, which have particulate-like nanostructures composed of reflectin proteins that scatter light. Typically, reflectins clump together and form the nanoparticles, so light isn't absorbed or directly transmitted; instead, the light scatters or bounces off of them, making the leucophores appear bright white.

"We wanted to engineer mammalian cells to stably, instead of temporarily, form reflectin nanostructures for which we could better control the scattering of light," says Gorodetsky. That's because if cells allow light through with little scattering, they'll seem more transparent. Alternatively, by scattering a lot more light, cells will become opaque and more apparent. "Then, at a cellular level, or even the culture level, we thought that we could predictably alter the cells' transparency relative to the surroundings or background," he says.

To change how light interacts with cultured cells, Georgii Bogdanov, a graduate student in Gorodetsky's lab who is presenting the results, introduced squid-derived genes that encoded for reflectin into human cells, which then used the DNA to produce the protein. "A key advance in our experiments was getting the cells to stably produce reflectin and form light-scattering nanostructures with relatively high refractive indices, which also allowed us to better image the cells in three dimensions," says Bogdanov.

In experiments, the team added salt to the cells' culture media and observed the reflectin proteins clumping together into nanostructures. By systematically increasing the salt concentration, Bogdanov got detailed, time-lapse 3D images of the nanostructures' properties. As the nanoparticles became larger, the amount of light that bounced off the cells increased, consequently tuning their opacity.

Then, the COVID-19 pandemic hit, leaving the researchers to wonder what they could do to advance their investigation without being physically in the lab. So, Bogdanov spent his time at home developing computational models that could predict a cell's expected light scattering and transparency before an experiment was even run. "It's a beautiful loop between theory and experiments, where you feed in design parameters for the reflectin nanostructures, get out specific predicted optical properties and then engineer the cells more efficiently -- for whatever light-scattering properties you might be interested in," explains Gorodetsky.

On a basic level, Gorodetsky suggests that these results will help scientists better understand squid skin cells, which haven't been successfully cultured in a laboratory setting. For example, previous researchers postulated that reflectin nanoparticles disassemble and reassemble to change the transparency of tunable squid leucophores. And now Gorodetsky's team has shown that similar rearrangements occurred in their stable engineered mammalian cells with simple changes in salt concentration, a mechanism that appears analogous to what has been observed in the tunable squid cells.

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Scientists imbue cells with pathway to make own drugs

Thank the rare crested ibis for a clue that could someday help our bodies make better drugs.

The species of bird is the only one known to naturally produce an enzyme able to generate a noncanonical amino acid; that is, one not among the 20 necessary to encode most proteins.

That it exists -- a discovery made through computational comparison of genome databases -- proves it's possible for that enzyme to work within the context of living cells, even if scientists don't know what it does for the bird.

But they have a pretty good idea of what it could do for us.

A new study by Rice University chemist Han Xiao, theoretical physicist Peter Wolynes and their colleagues shows that amino acid, sulfotyrosine (sTyr), a mutant of the standard amino acid tyrosine, is a key building block to program living cells that express therapeutic proteins. It could potentially allow cells to serve as sensors that monitor their environments and respond with the necessary treatment.

Mimicking the ibis' ability to synthesize sTyr and incorporate it into proteins requires modifying a cell's DNA with a mutant codon that, in turn, makes the transferase enzyme, sulfotransferase 1C1, found in the bird. This catalyzes the generation of sTyr, an essential recognition moiety in a variety of biomolecular interactions.

The proof-of-concept study produced for the first time mammalian cells that synthesize sTyr. In an experiment, the Xiao lab made cells that enhanced the potency of thrombin inhibitors, anticoagulants used to prevent blood clotting.

The study appears in Nature Communications.

"In nature, most of our species are made with 20 canonical building blocks," Xiao said. "If you want to add an additional building block, you need to think about how to make it. We solved that problem: We can ask the cell to make it.

"But then we have to have the translational machinery to recognize it. And a special codon to encode this new building block," he said. "With this study, we've fulfilled all three of these requirements."

Xiao received a National Institutes of Health grant in 2019 to see if cells could be programmed to make substances with extra amino acids. The new study demonstrates the lab's dramatic progress.

Up to now, scientists would feed chemically synthesized noncanonical amino acids into cells. Having the cell do the work is far more efficient, Xiao said, but that requires the discovery of a new transferase enzyme with tyrosine pockets that could bind sulfate. That lock-and-key combination could then be used as the foundation for a variety of catalysts.

"Now, through this new strategy to modify proteins, we can totally change a protein's structure and its function," he said. "For our thrombin inhibitors models, we showed that putting an unnatural building block in the drug can make the drug much more potent."

It was worth a look to see if nature had beaten them to a useful codon. For that, Xiao enlisted Wolynes, co-director of the Center for Theoretical Biological Physics, whose lab compared genome databases and found sulfotransferase 1C1 in the ibis.

The Xiao lab employed a mutant amber stop codon, a three-nucleotide group of uracil, adenine and guanine, to encode the desired sulfotransferase, resulting in a completely autonomous mammalian cell line capable of biosynthesizing sTyr and incorporating it with great precision into proteins.

"We got lucky," Xiao said. "Ibis is the only species doing this, which was discovered by a sequence similarity search of genomic information. After that, we asked if they can figure out why this enzyme recognizes tyrosine but our human sulfotransferase cannot."

The Wolynes team employed AlphaFold2, an artificial intelligence program developed by Alphabet/Google's DeepMind that predicts proteins structures.

The researchers expect to use the combination of bioinformatics and computationally enhanced screening to produce a library of biosynthesized noncanonical amino acids.

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