Abstract
The avian-origin influenza A virus polymerase is restricted in human cells. This restriction has been associated with species differences in host factor ANP32A. Avian ANP32A supports the activity of an avian-origin polymerase. However, the avian-origin polymerase is incompatible with human ANP32A. Avian ANP32A proteins harbor an additional 33 amino acids compared to human ANP32A proteins, which are crucial for their ability to support the avian-origin influenza virus polymerase. Here, we elucidate the interactions between ANP32A proteins and the influenza A virus polymerase using split luciferase complementation assays, coimmunoprecipitation, and in situ split Venus interaction assays. We show greater interaction of chicken ANP32A than human ANP32A with the viral polymerase and visualize these interactions in situ in the cell nucleus. We demonstrate that the 33 amino acids of chicken ANP32A and the PB2 627 domain of viral polymerase complex both contribute to this enhanced interaction. Finally, we show how these interactions are affected by the presence of viral RNA and the processivity of the polymerase, giving insights into the way that ANP32A might act during virus infection.IMPORTANCE Successful zoonotic transmission of influenza A virus into humans can lead to pandemics in an immunologically naive population. Host-encoded ANP32A proteins are required to support influenza A virus polymerase activity, and species differences in ANP32A can restrict the host range of influenza virus. Understanding how ANP32A proteins support the viral polymerase and how differences in ANP32A affect the ability of the polymerase to coopt these proteins will enhance our understanding of viral replication and species restriction as well as suggesting targeted antiviral approaches to treat influenza virus infection.
Highlights
The avian-origin influenza A virus polymerase is restricted in human cells
We chose to develop the assay using the construct that fused the N terminus of Gaussia luciferase onto the C terminus of PB1 and the C terminus of Gaussia luciferase onto the C terminus of chANP32A, since this combination gave the highest luciferase signal, likely because it allowed a sterically optimal alignment of the luciferase fragments (Fig. 1c)
We show greater binding of chANP32A than huANP32A with the Influenza A virus (IAV) polymerase and demonstrate that this is due to interactions involving the 33 amino acids in chANP32A and the 627 domain of PB2
Summary
The avian-origin influenza A virus polymerase is restricted in human cells This restriction has been associated with species differences in host factor ANP32A. Avian ANP32A proteins harbor an additional 33 amino acids compared to human ANP32A proteins, which are crucial for their ability to support the avian-origin influenza virus polymerase. Host-encoded ANP32A proteins are required to support influenza A virus polymerase activity, and species differences in ANP32A can restrict the host range of influenza virus. To be capable of replication and efficient transmission between humans, the virus must undergo adaptions to overcome the host range barriers present in the new host These barriers exist at several points during the viral replication cycle, including replication of the viral genome by the IAV RNA polymerase [1]. Since amino acid 627 of PB2 lies on a solvent-exposed region of the polymerase, it is accessible to host factors, and it is likely that differences in factors in different hosts drive
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