Structures of the Tilapia Lake Virus replication complex show that ANP32 is a host factor for replication across the Articulavirales order

Abstract

Tilapia Lake Virus (TiLV) is a species in the Amnoonviridae family within the Articulavirales order of nuclear-replicating, segmented, negative-strand RNA viruses. This order also encompasses the well-known, but phylogenetically distant Orthomyxoviridae family that includes influenza and Thogoto viruses. Influenza virus replication, but not Thogoto virus, essentially relies on the host acidic nuclear phosphoprotein 32 (ANP32) to form a functional genome replication complex, which comprises an asymmetric viral polymerase dimer composed of a replicase bridged by ANP32 to an encapsidase. However, remains unclear whether this dependency is conserved in divergent viruses from the same order. Using in vitro reconstitution, biophysical and biochemical analyses and high-resolution structure determination by single-particle cryo-electron microscopy, we investigate the complexes formed between the TiLV RNA-dependent RNA polymerase (TiLV-Pol) and either tilapia (ti) or human (hu) ANP32A. We show that the tiANP32 leucine-rich repeat domain acts as a scaffold that stabilizes apo-TiLV-Pol in an encapsidase conformation. The tiANP32-encapsidase complex is then able to recruit and stabilise a second TiLV-Pol in a replicase conformation. The tiANP32-stabilised asymmetric TiLV-Pol dimer has an architecture that is remarkably similar to its orthomyxovirus replication complex counterpart, despite the 40% smaller size of TiLV-Pol. We also demonstrate that the ANP32 low-complexity acidic region interacts with the TiLV nucleoprotein, consistent with a conserved role in co-ordinating ribonucleoprotein assembly. Overall, our findings suggest that ANP32 is an ancient and likely broadly conserved host factor for viral genome replication and encapsidation across the Articulavirales order. Furthermore, TiLV is an emerging fish pathogen causing high mortality in aquaculture and our results provide insight into tilapia ANP32 modifications that could be used to engineer TiLV-resistant tilapia. AUTHOR SUMMARY The Articulavirales order of nuclear-replicating, segmented, negative-strand RNA viruses includes both the well-known Orthomyxoviruses such as influenza and Thogoto viruses and the recently discovered and distantly related Amnoonviridae family of mainly fish viruses, such as Tilapia Lake Virus (TiLV). It is thought that most orthomyxoviruses depend on host factor acidic nuclear phosphoprotein 32 (ANP32) to form the active genome replication complex. This comprises an asymmetric dimer of two viral polymerases (a replicase and an encapsidase) bridged by ANP32. Using in vitro biochemical, biophysical and high-resolution structure-determination methods, we investigate whether TiLV also requires host ANP32 for replication. We find that TiLV polymerase (TiLV-Pol) interacts with tilapia ANP32A (tiANP32A) to form an asymmetric dimer that architecturally resembles the influenza virus replication complex, despite TiLV-Pol being only 60% of the size of influenza polymerase and completely diverged in sequence. Furthermore, the C-terminal acidic tail of ANP32 binds TiLV nucleoprotein, suggesting that ANP32 could also be important for progeny genome encapsidation. These finding suggests that ANP32 is an ancient and likely broadly conserved host factor for viral replication across the Articulavirales order. Our results also show how genetic engineering of fish ANP32 could potentially be used to combat economically damaging fish diseases.