New insights into how bird flu crosses the species barrier

The Cusack group at EMBL Grenoble studies the influenza virus replication process, and their new research uncovers the different mutations that occur in avian influenza viruses to enable them to replicate inside mammalian cells.

Some strains of avian influenza can cause severe illness and death. Fortunately, there are significant biological differences between birds and mammals, so avian influenza does not usually spread from birds to other species. To infect mammals, avian influenza viruses must mutate to overcome two main barriers: the ability to enter cells and the ability to replicate within those cells. To cause an epidemic or pandemic, they must also acquire the ability to transmit between humans.

However, sporadic infections of wild and domestic mammals with avian influenza are becoming increasingly common. Of particular concern is the recent unexpected infection of dairy cows in the United States with an avian influenza H5N1 strain, which may become endemic in cattle. This may facilitate adaptation to humans, and several cases of human infection have been reported, although so far the symptoms have been mild.

At the heart of this process is polymerase, the enzyme that orchestrates viral replication inside the host cell. This flexible protein can rearrange itself for the different functions it performs during infection. These functions include transcription (copying viral RNA into messenger RNA to create viral proteins) and replication (making copies of viral RNA and packaging it into new viruses).

Viral replication is a complex process to study because it involves two viral polymerases and the host cell protein ANP32. These three proteins form the replication complex, the molecular machine that carries out the replication. ANP32 is known as a “chaperone” and acts as a stabilizer for certain cellular proteins thanks to a key structure: its long acidic tail. In 2015, it was discovered that ANP32 is essential for influenza virus replication, but its function was not fully understood.

The results of a new study published in the journal Nature Communications, ANP32 has been shown to function as a bridge between the two viral polymerases (termed Replicase and Encapsidase. These names reflect the two different structures that the polymerase adopts to perform two distinct functions: making copies of the viral RNA (Replicase) and encase the copy in a protective coating with the help of ANP32 (Encapsidase).

ANP32 acts as a stabilizer of the replication complex through its tail, allowing the formation of the replication complex in the host cell. Interestingly, the tail of ANP32 differs between birds and mammals, but the core of the protein is very similar. This biological difference explains why avian influenza viruses do not easily replicate in mammals or humans.

“The main difference between avian and human ANP32 is the 33 amino acid insertion in the avian tail, and the polymerase has to adapt to this difference,” explains Benoît Arragain, a postdoctoral researcher in Cusack's group and first author on the paper. “To replicate in human cells, the avian-adapted polymerase needs to acquire specific mutations that allow it to use human ANP32.”

To gain a deeper understanding of this process, Aragáin and collaborators obtained conformational structures of the replicase and encapsidase of human-adapted avian influenza polymerase (from the H7N9 strain) interacting with human ANP32, providing detailed information about amino acids that are important for the formation of the replication complex and the mutations that allow avian influenza polymerase to adapt to mammalian cells.

To obtain these results, Allagain In vitro They carried out their experiments at EMBL in Grenoble, using the Eukaryotic Expression Facility, the ISBG Biophysics Platform and a cryo-electron microscopy platform available through the Structural Biology Partnership. “We also collaborated with the Naffakh group at the Institut Pasteur, who performed cell experiments,” Arragain added. “In addition, we obtained a structure of the human influenza B replication complex, which is similar to that of influenza A. The cell 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 avian influenza viruses, and therefore the structure obtained from the H7N9 strain can be used and adapted to other strains, such as H5N1.

“We need to take seriously the threat of a new pandemic caused by a highly pathogenic, human-adapted and potentially lethal avian influenza strain,” said Steven Cusack, a senior scientist at EMBL Grenoble who led the study and has studied influenza viruses for 30 years. “One important response to this threat is to monitor virus mutations in the field. Knowing this structure allows us to interpret these mutations and assess whether strains are adapted to infect and be transmitted between mammals.”

These results are also useful for the long-term perspective of anti-influenza drug development, because there are no existing drugs that specifically target the replication complex. “But this is just the beginning,” said Kassak. “What we want to do next is understand how the replication complex works dynamically, in other words, get a more detailed picture of how it actively replicates.” The research group has already successfully conducted a similar study on the role that influenza polymerase plays in the viral transcription process.