Packaging of the Influenza Virus Genome Is Governed by a Plastic Network of RNA- and Nucleoprotein-Mediated Interactions

Abstract

The genome of influenza A virus is organized into eight ribonucleoproteins, each composed of a distinct RNA segment bound by the viral polymerase and oligomeric viral nucleoprotein. Packaging sequences unique to each RNA segment together with specific nucleoprotein amino acids are thought to ensure the precise incorporation of these eight ribonucleoproteins into single virus particles, and yet the underlying interaction network remains largely unexplored. We show here that the genome packaging mechanism of an H7N7 subtype influenza A virus widely tolerates the mutation of individual packaging sequences in three different RNA segments. However, combinations of these modified RNA segments cause distinct genome packaging defects, marked by the absence of specific RNA segment subsets from the viral particles. Furthermore, we find that combining a single mutated packaging sequence with sets of specific nucleoprotein amino acid substitutions greatly impairs the viral genome packaging process. Along with previous reports, our data propose that influenza A virus uses a redundant and plastic network of RNA-RNA and potentially RNA-nucleoprotein interactions to coordinately incorporate its segmented genome into virions. IMPORTANCE The genome of influenza A virus is organized into eight viral ribonucleoproteins (vRNPs); this provides evolutionary advantages but complicates genome packaging. Although it has been shown that RNA packaging sequences and specific amino acids in the viral nucleoprotein (NP), both components of each vRNP, ensure selective packaging of one copy of each vRNP per virus particle, the required RNA-RNA and RNA-NP interactions remain largely elusive. We identified that the genome packaging mechanism tolerates the mutation of certain individual RNA packaging sequences, while their combined mutation provokes distinct genome packaging defects. Moreover, we found that seven specific amino acid substitutions in NP impair the function of RNA packaging sequences and that this defect is partially restored by another NP amino acid change. Collectively, our data indicate that packaging of the influenza A virus genome is controlled by a redundant and plastic network of RNA/protein interactions, which may facilitate natural reassortment processes.

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

“…argues against a strict, non-variable sequence of vRNP interactions consistent with recent experimental and modelling reports 14,37,38 . In the latter case, we would have expected a single distinct MSC composition to stand out per MSC rank.…”

“…Nakatsu et al 39 applied scanning electron microscopy tomography to diverse influenza A and B strains and showed that at least 80% and in some cases even 100% of virions contained a complete genome arranged in the '7 + 1' pattern. Reverse genetics approaches have shown that deletions or mutations in a packaging signal sequence of a single vRNA can prevent the incorporation of other vRNPs into virions or virus-like particles 2,14,40 . Very likely, the dense arrangement of vRNPs in high-rank MSCs forms a surface favouring recognition and preferential incorporation of such complexes into assembling viruses.…”

“…The random model presumes that vRNP segments interact in a stochastic manner, leading to bundles and virions that do not necessarily contain a complete set of vRNPs 9 , and may contain multiple copies of one segment. However, recent reports employing different techniques [10][11][12][13][14][15] provide solid evidence for the selective packaging model: the different vRNPs interact specifically with each other and thus ensure the incorporation of exactly one copy of each segment into multi-segment complexes (MSC). Using length as a parameter to distinguish between vRNP species, electron tomography of intact virions 10,12 suggests that the viral genome is organised as an MSC with eight different segments, 7 of which are arranged around a central segment in a '7 + 1' pattern 2,16 .…”

Bats reveal the true power of influenza A virus adaptability

“…These comprise for example the co-selection of genome packaging signals in conjunction with a specific set (code) of amino acids in the viral nucleoprotein (NP) [19][20][21]. Both the segmentspecific packaging signals and a compatible NP amino acid code are compulsory to orchestrate the incorporation of the viral genome into progeny virions [22]. Therefore, because of nonmatching packaging sequences and a different NP amino acid code, New World bat and conventional non-bat IAVs are unable to reassort their genomes [20]-an additional unique feature of New World bat IAVs, making them a truly divergent entity of IAVs.…”

Frustration and Fidelity in Influenza Genome Assembly

“…Consistent with this idea, mutations in the conserved RNA terminal regions cause defects in genome packaging [12]- [16]. Genomic RNA strands are seen to physically bind in vitro and in vivo [17]- [19] via RNA base-pairing interactions [20]- [22] and interactions mediated by vRNP-associated proteins [23]- [25]. In addition to the equilibrium RNA-RNA binding measured in vitro, livecell imaging reveals that some inter-segment interactions are irreversible on the timescale of an infection [26].…”