Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization

Majarian, Timothy D.; Murphy, Robert F.; Lakdawala, Seema S.

doi:10.1371/journal.pcbi.1006199

Cited by 13 publications

(15 citation statements)

References 37 publications

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“…A striking finding was that for any given intermediate rank, there was not a single specific segment composition of high abundance, but several, more quantitative than qualitatively preferred, partly overlapping segment combinations. This observation indicates a limited variability of specific interactions between the vRNPs, but 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.…”

Section: Resultssupporting

confidence: 87%

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

et al. 2020

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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 × 10 5 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.

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Section: Resultssupporting

confidence: 87%

“…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.…”

Section: Resultssupporting

confidence: 81%

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

et al. 2020

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“…To further examine a potential packaging defect for the chimeric WSN [pdm-NS 5′] mutant, we performed multi-color fluorescence in situ hybridization (FISH) to assess segment colocalization during viral infection. We focused on the colocalization of the NS with the M segment, as our previous studies have shown that the intracellular distribution of the M segment is highly correlated with the distribution of the NS segment, and that these two segments cluster together in putative vRNA segment assembly network constructions with machine learning [27]. Host cells infected with either wildtype or chimeric mutant virus for both WSN and H1N1pdm backgrounds were fixed and stained with probes at 8 hpi ( Figure 5A ).…”

Section: Resultsmentioning

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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“…2). This work has led to the concept that IAV packaging is mediated by RNA:RNA interactions, a hypothesis that has been corrob-orated through more current methodologies (Dadonaite et al 2019;Majarian et al 2019). This knowledge of packaging requirements enabled one to build viral recombinants expressing a foreign segment and complement the missing viral protein using stably expressing cell lines (Shinya et al 2004;Martínez-Sobrido et al 2010;Ozawa et al 2011).…”

Section: Defining Iav Circuitrymentioning

confidence: 99%

Synthetic Virology: Building Viruses to Better Understand Them

tenOever

2019

Cold Spring Harb Perspect Med

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Generally comprised of less than a dozen components, RNA viruses can be viewed as well-designed genetic circuits optimized to replicate and spread within a given host. Understanding the molecular design that enables this activity not only allows one to disrupt these circuits to study their biology, but it provides a reprogramming framework to achieve novel outputs. Recent advances have enabled a "learning by building" approach to better understand virus biology and create valuable tools. Below is a summary of how modifying the preexisting genetic framework of influenza A virus has been used to track viral movement, understand virus replication, and identify host factors that engage this viral circuitry.

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Learning the sequence of influenza A genome assembly during viral replication using point process models and fluorescence in situ hybridization

Cited by 13 publications

References 37 publications

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

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

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

Synthetic Virology: Building Viruses to Better Understand Them

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