Visualising and quantifying the RNA products of influenza A virus polymerase for insights into regulation and host restriction

Abstract

Host restriction limits the emergence of pandemic strains from the influenza A virus avian reservoir. One important host range barrier is the RNA-dependent RNA polymerase protein (FluPol), which must adapt to undergo efficient replication within the mammalian nucleus. The restriction of avian FluPol in mammalian cells is believed to occur due to incompatibilities with mammalian orthologues of the host factor ANP32, which lack a 33 amino acid insertion relative to avian ANP32A. The quintessential mammalian-adapting mutation that overcomes this restriction is a single amino acid change from a glutamic acid to a lysine at position 627 on the PB2 protein of the FluPol (PB2 E627K).

In this project, we set out to establish an approach for visualising the RNA products of FluPol within the nucleus. We developed a strand-specific RNA fluorescence in situ hybridisation (RNA FISH) approach, using the commercial RNAscope assay. Subsequently, this protocol was adapted for correlative light and electron microscopy (CLEM) in collaboration with the electron microscopy platform at the Crick Institute. Simultaneously, we used RNAscope and RT-qPCR to investigate the mechanism by which ANP32 supports viral replication, and functions in host restriction. We discovered that FluPol requires ANP32 proteins to support both steps of genome replication: cRNA and vRNA synthesis. However avian FluPol (PB2 627E) is only restricted in the second step, vRNA synthesis, in mammalian cells. This suggests ANP32 may have an additional role in supporting vRNA synthesis, and it is this activity that is specifically blocked when avian influenza infects mammalian cells.

In summary, this study has established a strand-specific RNA FISH assay, suitable for dissecting the spatiotemporal details of IAV infection within the nucleus on a single-cell level. Moreover, this approach has been utilised, alongside RT-qPCR, to dissect the mechanism of the essential host factor ANP32. In combination, this has improved our understanding of the IAV polymerase, a major contributor to host restriction, and an important therapeutic target.