Insights in pro-influenza virus activity of ANP32 proteins

Abstract

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.