In recent years, public health, surveillance and vaccination measures have contributed to significant progress in reducing the impact of seasonal influenza epidemics caused by human influenza viruses A and B. A possible outbreak of avian influenza A (commonly known as ‘bird flu’) 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 flu virus can undergo to replicate in mammalian cells.
Some bird flu strains can cause serious illness and death. Fortunately, significant biological differences between birds and mammals normally prevent avian flu from spreading from birds to other species. To infect mammals, the avian flu virus must mutate to overcome two major barriers: the ability to enter the cell and replicate within that cell. To cause an epidemic or pandemic, it must also acquire the ability to be transmitted between people.
However, sporadic infection 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 US by an avian H5N1 strain, which is at risk of becoming endemic in cattle. This could facilitate adaptation to humans, and indeed some cases of transmission to humans have been reported, which have so far produced only mild symptoms.
At the heart of this process is the polymerase, an enzyme that orchestrates the replication of the virus in host cells. This flexible protein can rearrange itself based 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 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 can do this thanks to an important structure: its long acidic tail. In 2015, ANP32 was discovered to be crucial for influenza virus replication, but its function was not 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 adopted by the polymerases to perform two different functions: creating copies of the viral RNA (replicase) and wrap the copy in a protective coating with the help of ANP32 (encapsidase).
Through its tail, ANP32 acts as a stabilizer for the replication complex, allowing it to form in 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 bird flu virus does not replicate easily in mammals and humans.
“The main difference between avian and human ANP32 is an insertion of 33 amino acids in the avian tail, and the polymerase must adapt to this difference,” explains Benoît Arragain, a postdoctoral researcher in the Cusack group and first author of the publication . . “In order for the avian-adapted polymerase to replicate in human cells, it must acquire certain mutations to utilize human ANP32.”
To better understand this process, Arragain and co-workers obtained the structure of the replicase and encapsidase conformations of a human-adapted avian flu polymerase (from strain H7N9) while interacting with human ANP32. This structure provides detailed information about which amino acids are important in forming the replication complex and which mutations can cause the avian 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 of the Institut Pasteur, which conducted cellular experiments,” Arragain added. “In addition, we obtained the structure of the human influenza replication complex type B, 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 must be taken seriously,” said Stephen Cusack, EMBL Grenoble Senior Scientist who led the study and has been studying influenza viruses for 30 years. year. “One of the most important responses to this threat is to monitor mutations in the virus in the field. Knowing this structure allows us to interpret these mutations and assess whether a strain is adapted to infect and transmit between mammals.”
These results are also useful in the long-term perspective of anti-influenza drug development, as there are no existing drugs that specifically target the replication complex. “But this is just the beginning,” Cusack said. “What we want to do next is understand how the replication complex works dynamically, in other words, to know 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.
