Mammalian ANP32A and ANP32B Proteins Drive Differential Polymerase Adaptations in Avian Influenza Virus.

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome.

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.

A natural variant in ANP32B impairs influenza virus replication in human cells.

Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.

ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells.

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.

Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation.

Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.

Avian Influenza A Virus Polymerase Can Utilize Human ANP32 Proteins To Support cRNA but Not vRNA Synthesis.

Host restriction limits the emergence of novel pandemic strains from the influenza A virus avian reservoir. For efficient replication in mammalian cells, the avian influenza RNA-dependent RNA polymerase must adapt to use human orthologues of the host factor ANP32, which lack a 33-amino-acid insertion relative to avian ANP32A. Here, we find that influenza polymerase requires ANP32 proteins to support both steps of genome replication: cRNA and vRNA synthesis. However, avian strains are only restricted in vRNA synthesis in human cells. Therefore, avian influenza polymerase can use human ANP32 orthologues to support cRNA synthesis, without acquiring mammalian adaptations. This implies a fundamental difference in the mechanism by which ANP32 proteins support cRNA versus vRNA synthesis. IMPORTANCE To infect humans and cause a pandemic, avian influenza must first adapt to use human versions of the proteins the virus hijacks for replication, instead of the avian orthologues found in bird cells. One critical host protein is ANP32. Understanding the details of how host proteins such as ANP32 support viral activity may allow the design of new antiviral strategies that disrupt these interactions. Here, we use cells that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps of influenza genome replication. Unexpectedly, however, we found that avian influenza can use human ANP32 proteins for the first step of replication, to copy a complementary strand, without adaptation but can only utilize avian ANP32 for the second step of replication that generates new genomes. This suggests ANP32 may have a distinct role in supporting the second step of replication, and it is this activity that is specifically blocked when avian influenza infects human cells.

An Influenza A virus can evolve to use human ANP32E through altering polymerase dimerization.

Human ANP32A and ANP32B are essential but redundant host factors for influenza virus genome replication. While most influenza viruses cannot replicate in edited human cells lacking both ANP32A and ANP32B, some strains exhibit limited growth. Here, we experimentally evolve such an influenza A virus in these edited cells and unexpectedly, after 2 passages, we observe robust viral growth. We find two mutations in different subunits of the influenza polymerase that enable the mutant virus to use a novel host factor, ANP32E, an alternative family member, which is unable to support the wild type polymerase. Both mutations reside in the symmetric dimer interface between two polymerase complexes and reduce polymerase dimerization. These mutations have previously been identified as adapting influenza viruses to mice. Indeed, the evolved virus gains the ability to use suboptimal mouse ANP32 proteins and becomes more virulent in mice. We identify further mutations in the symmetric dimer interface which we predict allow influenza to adapt to use suboptimal ANP32 proteins through a similar mechanism. Overall, our results suggest a balance between asymmetric and symmetric dimers of influenza virus polymerase that is influenced by the interaction between polymerase and ANP32 host proteins.

Creating resistance to avian influenza infection through genome editing of the ANP32 gene family.

Chickens genetically resistant to avian influenza could prevent future outbreaks. In chickens, influenza A virus (IAV) relies on host protein ANP32A. Here we use CRISPR/Cas9 to generate homozygous gene edited (GE) chickens containing two ANP32A amino acid substitutions that prevent viral polymerase interaction. After IAV challenge, 9/10 edited chickens remain uninfected. Challenge with a higher dose, however, led to breakthrough infections. Breakthrough IAV virus contained IAV polymerase gene mutations that conferred adaptation to the edited chicken ANP32A. Unexpectedly, this virus also replicated in chicken embryos edited to remove the entire ANP32A gene and instead co-opted alternative ANP32 protein family members, chicken ANP32B and ANP32E. Additional genome editing for removal of ANP32B and ANP32E eliminated all viral growth in chicken cells. Our data illustrate a first proof of concept step to generate IAV-resistant chickens and show that multiple genetic modifications will be required to curtail viral escape.

Host Determinants of Influenza RNA Synthesis.

Influenza viruses are a leading cause of seasonal and pandemic respiratory illness. Influenza is a negative-sense single-stranded RNA virus that encodes its own RNA-dependent RNA polymerase (RdRp) for nucleic acid synthesis. The RdRp catalyzes mRNA synthesis, as well as replication of the virus genome (viral RNA) through a complementary RNA intermediate. Virus propagation requires the generation of these RNA species in a controlled manner while competing heavily with the host cell for resources. Influenza virus appropriates host factors to enhance and regulate RdRp activity at every step of RNA synthesis. This review describes such host factors and summarizes our current understanding of the roles they play in viral synthesis of RNA.