Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication

Williams, Graham D.; Townsend, Dana; Wylie, Kristine M.; Kim, Preston J.; Amarasinghe, Gaya K.; Kutluay, Sebla B.; Boon, Adrianus C. M.

doi:10.1038/s41467-018-02886-w

Cited by 69 publications

(90 citation statements)

References 40 publications

(56 reference statements)

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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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“…A revised influenza virus genome architecture has recently been proposed, which suggests that NP binds vRNA in a non-uniform and non-random manner [18, 21], but how this apparent NP specificity is achieved remained unanswered. It was thus unclear whether nucleotide changes in vRNA would alter the NP binding profile, or whether the NP binding profile would remain static due to an as-yet unidentified mechanism that would maintain NP binding at the original positions in the vRNA.…”

Section: Discussionmentioning

confidence: 99%

“…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. Another study utilizing PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation) [20], a technically related version of HITS-CLIP that can also identify NP binding sites with nucleotide resolution, reached the same conclusion that NP binding is not pervasive throughout the segments inside infected host cells [21].…”

Section: Introductionmentioning

confidence: 99%

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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“…Our analysis and the work of others showed that the segment ends are highly conserved for all 8 segments [16][17][18][19][20][21] . The segment ends are potentially attractive regions for targeting with Cas13d because of the potential to inhibit a broad range of IAV strains and because of the previous evidence that interfering with the packaging of one segment encoded on the segment ends can decrease the packaging efficiency of other segments and overall virion packaging 18,[22][23][24][25] . Using sequence representation from ~140 different strains of influenza, we designed crRNAs that could be robust across as many different IAV strains as possible.…”

Section: Crispr Pac-man Is Able To Inhibit Iav Infection In Human Lunmentioning

confidence: 99%

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

Abbott

Dhamdhere

Liu

et al. 2020

Preprint

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The outbreak of the coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

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Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication

Cited by 69 publications

References 40 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

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

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