ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells

Abstract

ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human acidic nuclear phosphoproteins (ANPs) ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or ANP32B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues; murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can do so. Intriguingly, IBV polymerase is able to use murine Anp32A. We show, using a domain swap and point mutations, that the leucine-rich repeat (LRR) 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions. IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.

Swine ANP32A supports avian influenza virus polymerase

“…A further difference between the ANP32 proteins of different species is the level of redundancy in their ability to support influenza polymerase. In humans, two paralogues – ANP32A and ANP32B – are essential but redundant influenza polymerase cofactors (14, 15). In birds, only a single family member – ANP32A - supports influenza virus polymerase activity, as avian ANP32B proteins are not orthologous to mammalian ANP32B (11, 15, 16).…”

“…In birds, only a single family member – ANP32A - supports influenza virus polymerase activity, as avian ANP32B proteins are not orthologous to mammalian ANP32B (11, 15, 16). In mice, only ANP32B can support influenza A polymerase activity (14, 15). Neither avian nor mammalian ANP32E proteins have been shown to support influenza polymerase activity (14–16).…”

Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

“…Genome editing using CRISPR systems is a valuable research tool because it allows targeted 58 changes to genomes of species of interest. Therefore, CRISPR editing can be applied to test the 59 functional role of a particular gene or variant in a trait of interest, such as resistance to infection 60 (Staller et al 2019). This can be achieved by editing the target species' genome at a location that 61 will result in knockout of the target gene, or by introducing domains which will activate or repress 62 its expression (Gilbert et al 2014; Doudna and Charpentier 2014).…”

Molecular Basis of Host-Adaptation Interactions between Influenza Virus Polymerase PB2 Subunit and ANP32A

“…13 hANP32A lacks an insertion of 33 disordered residues compared to avANP32A, restricting avH5N1 polymerase activity in mammalian cells ( Figure 1A). This restriction is lifted by E627K mutation, suggesting an essential role for ANP32A through interaction with PB2, [14][15][16][17][18][19][20][21][22] although there are currently no molecular descriptions of these interactions. The interaction between ANP32 and influenza polymerase is critical in supporting IAV replication and is attracting increasingly intense interest.…”

“…The interaction between ANP32 and influenza polymerase is critical in supporting IAV replication and is attracting increasingly intense interest. [16][17][18] Recent studies also point to the importance of related members of the ANP32 family, in particular ANP32B, [19][20][21] as well as the role of surface residues in the folded leucine rich region (LRR) of ANP32A. 22 Conformationally, the 627-NLS region of PB2 exhibits intriguing behaviour.…”