Insights into species-specific regulation of ANP32A on the mammalian-restricted influenza virus polymerase activity

Abstract

The ANP32A is responsible for mammalian-restricted influenza virus polymerase activity. However, the mechanism of ANP32A modulation of polymerase activity remains poorly understood. Here, we report that chicken ANP32A (chANP32A)-X1 and-X2 stimulated mammalian-restricted PB2 627E polymerase activity in a dose-dependent manner. Distinct effects of ANP32A constructs suggested that the 180 VK 181 residues within chANP32A-X1 are necessary but not sufficient to stimulate PB2 627E polymerase activity. The PB2 N567D, T598V, A613V or F636L mutations promoted PB2 627E polymerase activity and chANP32A-X1 showed additive effects, providing further support that species-specific regulation of ANP32A might be only relevant with the PB2 E627K mutation. Rescue of cycloheximide-mediated inhibition showed that ANP32A is species-specific for modulation of vRNA but not mRNA and cRNA, demonstrating chANP32A-X1 compensated for defective cRNPs produced by PB2 627E virus in mammalian cells. The promoter mutations of cRNA enhanced the restriction of PB2 627E polymerase in mammalian cells, which could be restored by chANP32A-X1, indicating that ANP32A is likely to regulate the interaction of viral polymerase with RNA promoter. Coimmunoprecipitation showed that ANP32A did not affect the primary cRNPs assembly. We propose a model that chANP32A-X1 regulates PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.

Avian Influenza A Virus polymerase can utilise human ANP32 proteins to support cRNA but not vRNA synthesis

“…Our data suggests this mismatch between the huANP32 LCAR and FluPol 627E can be tolerated for cRNA synthesis. This conclusion aligns with the observation that addition of chANP32A to human cells does not stimulate FluPol 627E cRNA synthesis (26).…”

Host ANP32A mediates the assembly of the influenza virus replicase

“…Species-specific differences in ANP32 proteins underpin the poor activity of avian FluPol A in mammalian cells 4 . Amino acid differences important for FluPol activity have been identified in both ANP32A LRR and ANP32A LCAR , but a 33 amino acid insertion in the avian ANP32A LCAR when compared to mammalian ANP32A LCAR was found to be the most critical for the ability of avian ANP32A to support the activity of avian FluPol A 4 , 18 , 19 ( Fig. 3a ).…”

“…ANP32A contributes to the stabilisation of this dimer by bridging the two FluPol molecules. Several lines of evidence suggest that ANP32A is involved in cRNA to vRNA replication although there is also evidence in favour of a role during vRNA to cRNA replication 7 , 18 , 29 , 30 . In particular, avian polymerase in human cells is incapable of generating replication competent cRNPs, suggesting that cRNA encapsidation and hence the correct assembly of cRNPs might be affected 29 ; this is fully consistent with our data showing that mutations at the FluPol R FluPol E dimer interface interfere with cRNA encapsidation.…”

Development of a method for evaluating the mRNA transcription activity of influenza virus RNA-dependent RNA polymerase through real-time reverse transcription polymerase chain reaction

“…In the analysis of influenza RdRp activity using the dual-luciferase reporter assay, the 5’ and 3' untranslated regions of vRNA (the region recognized and bound by viral polymerase) were inserted into the luciferase reporter system to detect and quantify RdRp activity [ 28 – 32 ]. The advantage of the dual-luciferase reporter assay is that it is useful for high-throughput screening of many compounds.…”