The avian influenza virus must undergo mutations to break through the species barrier and successfully infect and replicate in mammalian cells. Researchers have recently uncovered the structure of the virus’s polymerase when it interacts with a key human protein necessary for the virus’s replication within host cells. This new structural information on the replication complex reveals essential mutations the avian influenza polymerase needs in order to adapt to mammals, including humans. Such findings can assist scientists in tracking the evolution and adaptability of bird flu strains, like H5N1 or H7N9, as they learn to infect different species.
In recent years, measures in public health such as vaccination and surveillance have significantly reduced the impact of seasonal flu cases caused by human influenza viruses A and B. However, the potential for avian influenza A (also known as ‘bird flu’) to spread to mammals, including humans, remains a serious public health concern.
The Cusack group at EMBL Grenoble is investigating how influenza viruses replicate. Their latest research sheds light on the various mutations that the avian influenza virus can experience to replicate in mammalian cells.
While some avian influenza strains can lead to severe illness and death, natural biological differences between birds and mammals typically prevent the virus from spreading to other species. For the avian influenza virus to infect mammals, it must first mutate to overcome two primary challenges: entering the host cell and replicating inside it. For the virus to trigger an epidemic or pandemic, it must also gain the ability to transfer between humans.
Nonetheless, sporadic cases of bird flu contaminating wild and domestic mammals are becoming more frequent. Notably, there have been concerning reports of an avian H5N1 strain infecting dairy cows in the USA, raising the risk of it becoming endemic in cattle. This scenario could aid in its adaptation to humans, with a few instances of human transmission noted so far, albeit with only mild symptoms.
Central to this replication process is the polymerase, an enzyme that manages the virus’s replication within host cells. This versatile protein can rearrange in structure to perform various functions during infection, which include transcription—copying viral RNA into messenger RNA for producing viral proteins—and replication—creating copies of viral RNA for packaging into new viruses.
Studying viral replication is quite complex due to the involvement of two viral polymerases and a host cell protein called ANP32. These three proteins combine to form a replication complex, which acts as a machinery carrying out replication. ANP32, known as a ‘chaperone’, plays a key role by stabilizing certain cellular proteins, thanks to its unique long acidic tail. Although its importance for influenza virus replication was recognized in 2015, its specific functions were not fully understood.
The new research published in the journal Nature Communications indicates that ANP32 serves as a connector between the two viral polymerases, referred to as replicase and encapsidase. These names describe the two distinct shapes that the polymerases adopt to perform different functions: replicase generates copies of viral RNA, while encapsidase assists in wrapping these copies in a protective shell with ANP32’s aid.
Through its tail, ANP32 helps stabilize the formation of the replication complex within the host cell. Interestingly, the tail of ANP32 varies between birds and mammals, while the core structure remains quite similar. This biological distinction explains the difficulty that the avian influenza virus has in replicating within mammals and humans.
“The major difference between avian and human ANP32 is a 33-amino-acid addition in the avian tail, and the polymerase must adapt to this variation,” explained Benoît Arragain, a postdoctoral fellow in the Cusack group and lead author of this research. “For the avian-adapted polymerase to work in human cells, it needs to acquire specific mutations to utilize human ANP32.”
To gain further insights into this process, Arragain and his team deciphered the structure of the replicase and encapsidase forms of a human-adapted avian influenza polymerase (from strain H7N9), as it interacted with human ANP32. This structure provides vital details about which amino acids are crucial in forming the replication complex and which mutations could allow the avian influenza polymerase to adjust to mammalian cells.
Using in vitro experiments at EMBL Grenoble, Arragain utilized resources from the Eukaryotic Expression Facility, the ISBG biophysical platform, and the cryo-electron microscopy services available through the Partnership for Structural Biology. “We also worked with the Naffakh group at the Institut Pasteur, who conducted cellular experiments,” Arragain noted. “Additionally, we obtained the structure of the replication complex for human type B influenza, which resembles that of influenza A. The cellular studies supported our structural findings.”
The new insights into the influenza replication complex can be applied to investigate polymerase mutations in other avian influenza virus strains as well. Consequently, the structure obtained from the H7N9 strain can potentially be adjusted for other strains like H5N1.
“We must take the risk of a new pandemic caused by highly pathogenic, human-adapted avian influenza strains with high mortality rates very seriously,” warned Stephen Cusack, senior scientist at EMBL Grenoble and study leader, who has dedicated 30 years to researching influenza viruses. “Key strategies in addressing this threat involve monitoring the virus for mutations in the wild. Understanding this structure helps us interpret these mutations and evaluate whether a strain is adapting to infect and spread among mammals.”
These findings also hold significance for the long-term development of anti-influenza medications, given that no targeted drugs for the replication complex currently exist. “This is just the beginning,” noted Cusack. “Our next aim is to comprehend how the replication complex functions dynamically, gaining deeper insights into its active replication processes.” The research group has already successfully undertaken similar investigations into the role of influenza polymerase in the viral transcription process.
