A conserved influenza A virus nucleoprotein code controls specific viral genome packaging.

Étori Aguiar Moreira,Martin Schwemmle,Seema Lakdawala,Anna Weber,Gert Zimmer,Adolfo García-Sastre,Sebastian Giese,Larissa Kolesnikova,Hardin Bolte,Mindaugas Juozapaitis,Martin Beer

doi:10.1038/ncomms12861

Abstract

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. How the packaging signals regulate the specific incorporation of each RNA segment into virions and whether other viral or host factors are involved in this process is unknown. Here, we show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments. This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). Replacement of conserved SC35M NP residues by those of HL17NL10 NP resulted in RNA packaging defective IAV. Surprisingly, substitution of these conserved SC35M amino acids with HL17NL10 NP residues led to IAV with altered packaging efficiencies for specific subsets of RNA segments. This suggests that NP harbours an amino acid code that dictates genome packaging into infectious virions.

Highlights

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences
Infection of MDCKII cells with rCH2 at a multiplicity of infection (MOI) of 0.001 resulted in a 35-fold reduction in viral titres 24 h post infection (h.p.i.) (Fig. 1d), indicating that the CH2 chimeric NP supported the generation of infectious IAV, the resulting rCH2 virus was attenuated in viral growth
The R31G mutation in NP7 (NP7 À R31G) was a reversion that almost restored the packaging efficiency to wt SC35M NP levels (Fig. 5d). Using this virus-like particle (VLP)-based packaging assay, we found that wt Bat NP in combination with the additional seven wt SC35M genome segments did not mediate efficient packaging of an incorporation signals (IS) þ bundling sequences (BS) À green fluorescent protein (GFP) reporter minigenome (Supplementary Fig. 8). These results strongly suggest that the attenuation of SC35M virus following the introduction of Bat NP-specific amino acids into SC35M NP is due to a defect in packaging of a full complement of eight viral RNA segments and that the incompatibility between human IAV and bat influenza A-like viruses might be a consequence of this packaging defect

Summary

Introduction

Packaging of the eight genomic RNA segments of influenza A viruses (IAV) into viral particles is coordinated by segment-specific packaging sequences. We show that distinct amino acids of the viral nucleoprotein (NP) are required for packaging of specific RNA segments This was determined by studying the NP of a bat influenza A-like virus, HL17NL10, in the context of a conventional IAV (SC35M). While the sequences in the non-coding regions of the RNA segments are important for the incorporation of the vRNPs into viral particles ( referred to as ‘incorporation signals’), the sequences in the 30 and 50 regions of the open reading frames (ORF) seem to be involved in the formation of the 7 þ 1 genome bundle ( referred to as ‘bundling signals’)[16]. Depending on which amino acids of bat NP are inserted, different sets of viral genome segments were preferentially incorporated into viral particles, suggesting that NPs of conventional IAV harbour a conserved amino acid code that, together with the eight segment-specific RNA packaging sequences, coordinate viral genome packaging

Methods

Results

Conclusion