Genome-wide analysis of influenza viral RNA and nucleoprotein association

Lee, Nara; Sage, Valerie Le; Nanni, Adalena V; Snyder, Dan; Cooper, Vaughn S.; Lakdawala, Seema S.

doi:10.1093/nar/gkx584

Cited by 82 publications

(126 citation statements)

References 54 publications

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“…The vRNP is responsible for transcription and replication of constituent vRNAs and the incorporation of vRNAs into progeny virions. Recent studies show that NPs, in the context of vRNPs, bind to vRNAs nonuniformly without sequence specificity . Importantly, next‐generation sequencing analyses have demonstrated that some regions of the vRNAs, in the context of vRNPs, are free of NPs and able to form secondary or tertiary structures on the surface of the rod‐shaped vRNPs …”

Section: Introductionmentioning

confidence: 99%

“…Recent studies show that NPs, in the context of vRNPs, bind to vRNAs nonuniformly without sequence specificity. 1,2 Importantly, next-generation sequencing analyses have demonstrated that some regions of the vRNAs, in the context of vRNPs, are free of NPs and able to form secondary or tertiary structures on the surface of the rod-shaped vRNPs. 3 It has been demonstrated that progeny virions selectively package a single copy of each of the eight vRNAs.…”

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confidence: 99%

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In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

Miyamoto

Noda

2020

Microbiology and Immunology

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The genome of influenza A virus consists of eight‐segmented, single‐stranded, negative‐sense viral RNAs (vRNAs). Each vRNA contains a central coding region that is flanked by noncoding regions. It has been shown that upon virion formation, the eight vRNAs are selectively packaged into progeny virions through segment‐specific packaging signals that are located in both the terminal coding regions and adjacent noncoding regions of each vRNA. Although recent studies using next‐generation sequencing suggest that multiple intersegment interactions are involved in genome packaging, contributions of the packaging signals to the intersegment interactions are not fully understood. Herein, using synthesized full‐length vRNAs of H1N1 WSN (A/WSN/33 [H1N1]) virus and short vRNAs containing the packaging signal sequences, we performed in vitro RNA binding assays and identified 15 intersegment interactions among eight vRNAs, most of which were mediated by the 3′‐ and 5′‐terminal regions. Interestingly, all eight vRNAs interacted with multiple other vRNAs, in that some bound to different vRNAs through their respective 3′‐ and 5′‐terminal regions. These in vitro findings would be of use in future studies of in vivo vRNA–vRNA interactions during selective genome packaging.

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

Miyamoto

Noda

2020

Microbiology and Immunology

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“…All eight IAV segments are coated by viral nucleoprotein (NP) molecules, which until recently were thought to cover the entire length of the segments uniformly like ‘beads on a string’ [12–17]. Using HITS-CLIP (high throughput sequencing of RNA isolated by crosslinking and immunoprecipitation), we have previously demonstrated that NP binding to vRNA in virions is not regular but enriched at some regions of the segments while depleted at others, providing evidence for an alternative model of non-uniform NP association to vRNA [18]. The advantage of HITS-CLIP is that, unlike other versions of the CLIP methodology, such as iCLIP or eCLIP [19], it uncovers the entire footprint of vRNA protected by NP rather than focusing on the NP-crosslinked sites on vRNA.…”

Section: Introductionmentioning

confidence: 99%

“…We have further shown that the NP binding profiles of strains with a high degree of nucleotide sequence conservation can differ markedly [18]. The A/WSN/1933 (WSN) and A/California/07/2009 (H1N1pdm) strains, both of the H1N1 subtype with an overall sequence homology of 85%, contain NP binding sites that are shared between both strains as well as unique to each strain.…”

Section: Introductionmentioning

confidence: 99%

“…Previous in vitro binding assays indicated that NP binds RNA in a sequence-independent manner [22, 23], raising the question of how strain-specific NP binding to vRNA is accomplished. We did observe a statistically robust bias for NP binding sites in that they are relatively depleted in uracils and enriched for guanines compared to genome-wide nucleotide content [18]. However, given the vast spread in nucleotide content among all NP peaks in the viral genome, this bias is unlikely to be the sole underlying determining factor of NP recruitment.…”

Section: Introductionmentioning

confidence: 99%

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Altered Nucleoprotein Binding to Influenza Virus RNA Impacts Packaging Efficiency and Replication

Sage

Kanarek

Nturibi

et al. 2019

Preprint

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24The genome of Influenza A viruses consists of eight negative-sense RNA segments that 25 are bound by viral nucleoprotein (NP). We recently showed that NP binding is not uniform along 26 the segments but exhibits regions of enrichment as well as depletion. Furthermore, genome-wide 27 NP binding profiles are distinct even in strains with high sequence similarity, such as the two 28 H1N1 strains A/WSN/1933 and A/California/07/2009. Here, we performed interstrain segment 29 swapping experiments with segments of either high or low congruency in NP binding, which 30 suggested that a segment with a similar overall NP binding profile preserved replication fitness of 31 the resulting virus. Further sub-segmental swapping experiments demonstrated that NP binding 32 is affected by changes to the underlying nucleotide sequence, as NP peaks can either become 33 lost or appear de novo at mutated regions. Unexpectedly, these local nucleotide changes in one 34 segment not only affect NP binding in cis, but also impact the genome-wide NP binding profile on 35 other segments in a vRNA sequence-independent manner, suggesting that primary sequence 36 alone is not the sole determinant for NP association to vRNA. Moreover, we observed that sub-37 segmental mutations that affect NP binding profiles can result in reduced replication fitness, which 38 is caused by defects in vRNA packaging efficiency and an increase in semi-infectious particle 39 production. Taken together, our results indicate that the pattern of NP binding to vRNA is 40 important for efficient virus replication. 41 3 Author Summary 42 Each viral RNA (vRNA) segment is bound by the polymerase complex at the 5′ and 3′ 43 ends, while the remainder of the vRNA is coated non-uniformly and non-randomly by 44 nucleoprotein (NP). To explore the constraints of NP binding to vRNA, we used high-throughput 45 sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of mutant H1N1 46 strains with exchanged vRNA sequences and observed that NP binding can be changed based 47 on vRNA sequence. The most striking observation of our study is that nucleotide changes in one 48 segment can have genome-wide effects on the NP binding profile of other segments. We refer to 49 this phenomenon as the 'butterfly effect' of influenza packaging. Our results provide an important 50 context in which to consider future studies regarding influenza packaging and assembly.51 4 Introduction 52The segmented nature of influenza A virus (IAV) genomes poses a logistical challenge for 53 viral replication, as all of the eight negative-sense single-stranded RNA segments must find their 54 way into a budding virion to give rise to an infectious particle [1, 2]. Following nuclear export, viral 55 ribonucleoprotein complexes (vRNP) containing newly synthesized viral RNA (vRNA) assemble 56 on recycling endosomes en route to the plasma membrane for packaging into virions [3, 4]. An 57 accumulating body of evidence suggests that the intracellular pre-assembly process of vRNP 58 trafficking is media...

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Local changes in viral RNA sequence drive global changes in influenza nucleoprotein binding

Sage

Kanarek

Lakdawala

et al. 2023

Journal of Medical Virology

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The genome of influenza A viruses (IAV) consists of eight negative-sense RNA segments that are coated by viral nucleoprotein (NP). Until recently, it was assumed that NP binds viral genomic RNA (vRNA) uniformly along the entire segment. However, genome-wide studies have revised the original model in that NP instead binds preferentially to certain regions of vRNA, while others are depleted for NP binding. Even strains with high sequence similarity exhibit distinct NP-binding profiles. Thus, it remains unknown how NP-binding specificity to vRNA is established. Here we introduced nucleotide changes to vRNA to examine whether

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Genome-wide analysis of influenza viral RNA and nucleoprotein association

Cited by 82 publications

References 54 publications

In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

In vitro vRNA–vRNA interactions in the H1N1 influenza A virus genome

Altered Nucleoprotein Binding to Influenza Virus RNA Impacts Packaging Efficiency and Replication

Local changes in viral RNA sequence drive global changes in influenza nucleoprotein binding

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