Selective flexible packaging pathways of the segmented genome of influenza A virus

Ivan Haralampiev,Simon Prisner,Matthias Schade,Mor Nitzan,Fabian Jolmes,Nir Friedman,Jasmine Chamiolo,Andreas Herrmann,Niklaas Nilson,Franziska Winter,Oliver Seitz,Kalle Jongen,Max Schreiber,Maria Loidolt-Krüger,Thorsten Wolff

doi:10.1038/s41467-020-18108-1

Abstract

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 105 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.

Highlights

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins that consist of negative sense viral RNA covered by the IAV nucleoprotein
The IAV genome is encoded in eight negatively orientated viral RNAs ranging from 0.9 to 2.3 kb in length. They are organised as viral ribonucleoproteins, which are decorated with viral nucleoproteins (NP) and a single viral RNA-dependent RNA polymerase (RdRp)[2]
Analysis of the segment composition of intermediate multi-segment complexes (MSC) in infected cells is indicative of a flexible selective packaging mechanism by which viral ribonucleoproteins (vRNPs) are assembled into a complete octameric IAV genome complex

Summary

Introduction

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Recent reports employing different techniques[10,11,12,13,14,15] provide solid evidence for the selective packaging model: the different vRNPs interact with each other and ensure the incorporation of exactly one copy of each segment into multi-segment complexes (MSC). There is broad consensus on the non-random, selective packaging model of viral genome formation, the assembly pathway(s), i.e., the order in which segments form a complete octameric MSC, has not yet been elucidated[8]. Using spinning-disc microscopy to detect vRNA spots and their colocalisation, we are able to extract the segment composition of about 105 MSCs. Analysis of the segment composition of intermediate MSCs in infected cells is indicative of a flexible selective packaging mechanism by which vRNPs are assembled into a complete octameric IAV genome complex. These results, together with the observed impairment of vRNP bundling upon non-permissive IAV infection, provide a framework for future work to decipher the precise rules of segment bundling and gene reassortment, and the involvement of host-cell factors

Methods

Results

Conclusion