New insights into how bird flu crosses species barriers

In recent years, public health measures, surveillance and vaccination have made significant progress in containing the impact of seasonal influenza 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 public health threat.

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

Some strains of avian influenza can cause severe illness and death. Fortunately, significant biological differences between birds and mammals usually prevent avian influenza from spreading from birds to other species. To infect mammals, the avian influenza virus must mutate and overcome two major barriers: the ability to enter the cell and to replicate in that cell. In addition, to cause an epidemic or pandemic, it must develop the ability to be transmitted between humans.

However, sporadic infection with avian influenza in wild and domesticated mammals is becoming increasingly common. Of particular concern is the recent unexpected infection of dairy cows in the United States with an H5N1 strain of avian influenza that may become endemic in cattle. This may facilitate adaptation to humans, and indeed a few cases of transmission to humans have been reported, causing only mild symptoms so far.

At the heart of this process is polymerase, an enzyme that controls the replication of the virus in host cells. This flexible protein can rearrange itself depending on 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 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,” which means it acts as a stabilizer for certain cellular proteins. It can do this thanks to a key structure – its long acidic tail. In 2015, ANP32 was discovered to be critical for influenza virus replication, but its function was not yet fully understood.

The results of the new study, published in the journal Nature communication, show that ANP32 acts as a bridge between the two viral polymerases – called replicase and encapsidase. The names reflect the two different conformations that the polymerases adopt to perform two different functions – making copies of the viral RNA (replicase) and packaging the copy in a protective shell with the help of ANP32 (encapsidase).

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

“The main difference between avian and human ANP32 is a 33-amino acid insertion in the bird's tail, and the polymerase must adapt to this difference,” explained Benoît Arragain, a postdoctoral fellow in the Cusack group and first author of the paper. “In order 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 determined 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 about which amino acids are important for the formation of the replication complex and which mutations might allow the avian influenza polymerase to adapt to mammalian cells.

To obtain these results, Arragain performed 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, which performed cellular experiments,” added Arragain. “In addition, we obtained the structure of the replication complex of human influenza type B, which is similar to that of influenza type A. The cellular experiments confirmed our structural data.”

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

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

These findings are also useful for the long-term development of flu drugs, since there are no drugs yet that specifically target the replication complex. “But this is just the beginning,” Cusack said. “Next, we want to understand how the replication complex works dynamically, that is, to know more precisely how it actively replicates.”

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