New insights into how bird flu crosses the species barrier

In recent years, public health measures, surveillance and vaccination have contributed to significant progress in reducing the impact of seasonal influenza epidemics, caused by human influenza A and B viruses. However, a potential 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 by this group highlights the different mutations that the avian influenza virus can undergo in order to replicate in mammalian cells.

Some strains of avian influenza can cause severe disease and high mortality. Fortunately, important 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 obstacles: the ability to enter and replicate within a cell. To cause an epidemic or pandemic, it must also acquire the ability to transmit between humans.

However, sporadic contamination of wild and domestic mammals with avian influenza is becoming increasingly common. Of particular concern is the unexpected infection of dairy cows in the United States with an avian strain of H5N1, which is likely to become endemic in cattle. This could 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 polymerase, an enzyme that orchestrates the replication of the virus in host cells. This flexible protein can rearrange itself to perform different functions during infection. These include transcription (copying viral RNA into messenger RNA to make viral proteins) and replication (making copies of viral RNA to package them 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 it acts as a stabilizer for certain cellular proteins. It is able to do this because of a key structure: its long acidic tail. In 2015, ANP32 was discovered to be essential for influenza virus replication, but its function was not fully understood.

The results of the new study, published in the journal Communication on nature, The results of this study show that ANP32 acts as a bridge between two viral polymerases, called replicase and encapsidase. These names reflect the two distinct conformations adopted by the polymerases to perform two different functions: creating copies of the viral RNA (replicase) and packaging the copy in a protective coating with the help of ANP32 (encapsidase).

Through its tail, ANP32 acts as a stabilizer of the replication complex, allowing it to form in the host cell. Interestingly, the tail of ANP32 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 ANP32 and human ANP32 is a 33-amino acid insertion in the bird’s tail, and the polymerase has to adapt to this difference,” says Benoît Arragain, a postdoctoral researcher in the Cusack group and first author of the paper. “For the avian-adapted polymerase to replicate in human cells, it has to 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 the H7N9 strain) as they interacted with human ANP32. This structure provides detailed information on the amino acids important in the formation of the replication complex and on the mutations that 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 biophysics platform and the cryo-electron microscopy platform available through the Partnership for Structural Biology.

“We also collaborated with the Naffakh group at the Pasteur Institute, which performed cellular experiments,” Arragain added. “In addition, we obtained the structure of the human influenza B replication complex, which is similar to that of influenza A. The cellular experiments confirmed our structural data.”

This new knowledge about the influenza replication complex can be used to study polymerase mutations in other similar strains of avian influenza virus. It is thus 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, and fatal avian influenza strains must be taken seriously,” said Stephen Cusack, a senior scientist at EMBL Grenoble who led the study and who has studied influenza viruses for 30 years.

“One of the main responses to this threat is to monitor virus mutations in the field. Knowing this structure allows us to interpret these mutations and assess whether a strain is on the path to adaptation to infect and transmit the virus between mammals.”

These findings are also useful in the long term for developing drugs against influenza, because there are no drugs that specifically target the replication complex. “But this is just the beginning,” Cusack said. “What we want to do now is understand how the replication complex works dynamically, in other words, learn in more detail how it actively carries out replication.”

The group has already successfully conducted similar studies on the role of influenza polymerase in the viral transcription process.