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

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

Swine ANP32A supports avian influenza virus polymerase

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as 'mixing vessels', being susceptible to both avian and human origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports activity of avian origin influenza virus polymerases, and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced pro-viral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the 'mixing vessel' trait of swine and further our understanding of the evolution and ecology of viruses in this host.Importance Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus 'mixing vessels'. In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this through binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as 'mixing vessels'

Insights in pro-influenza virus activity of ANP32 proteins

All viruses usurp the host machinery to assist their replication and influenza virus is no exception. One such host factor is the acidic nuclear phosphoprotein of 32 kilodaltons ANP32A and its closely related paralogue ANP32B. Taking a CRISPR/Cas9 genome editing approach (Chapter III) it was demonstrated that human ANP32A and ANP32B are a pair of functionally redundant essential host factors for influenza A and B virus polymerase (FluPol) activity and influenza A virus replication in human cells (Chapter IV). Ablation of either ANP32A or ANP32B has a minor effect on FluPol activity, but ablation of both paralogues leads to complete abrogation of FluPol activity and virus replication. Using these double knockout (dKO) cells it was shown that mouse ANP32A lacks proviral function due to a single amino acid substitution at position 130 (Chapter IV). Natural variation in the genes encoding ANP32A and ANP32B is investigated next (Chapter V). A missense single nucleotide variant (SNV) in the Anp32B gene codes for a mutant protein with alanine at position 130 instead of the wildtype aspartic acid (ANP32B-D130A). This variant is relatively common in carriers of Hispanic/Latino descent and it was hypothesised that carriers of this SNV may have some natural genetic protection against influenza virus. CRISPR/Cas9 editing in human cells recapitalised the homozygous mutant genotype and it was found that FluPol activity and virus replication were compromised in the presence of ANP32B-D130A. Crucially, ANP32B-D130A exerted a dominant-negative effect over wildtype ANP32B and moreover interfered with the functionally redundant paralogue ANP32A (Chapter V). Finally in Chapter VI mutational analysis was carried out in order to map the proviral activity of ANP32 proteins to further structural elements and domains.Open Acces