Dataset Information

Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network.

ABSTRACT: Selective assembly of influenza virus segments into virions is proposed to be mediated through intersegmental RNA-RNA interactions. Here, we developed a method called 2CIMPL that includes proximity ligation under native conditions to identify genome-wide RNA duplexes. Interactions between all eight segments were observed at multiple sites along a given segment and are concentrated at hotspots. Furthermore, synonymous nucleotide changes in a hotspot decreased the formation of RNA-RNA interactions at this site and resulted in a genome-wide rearrangement without a loss in replicative fitness. These results indicate that the viral RNA interaction network is flexible to account for nucleotide evolution. Moreover, comparative analysis of RNA-RNA interaction sites with viral nucleoprotein (NP) binding to the genome revealed that RNA junctions can also occur adjacent to NP peaks, suggesting that NP association does not exclude RNA duplex formation. Overall, 2CIMPL is a versatile technique to map in vivo RNA-RNA interactions.

SUBMITTER: Le Sage V

PROVIDER: S-EPMC7372595 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network.

Le Sage Valerie V Kanarek Jack P JP Snyder Dan J DJ Cooper Vaughn S VS Lakdawala Seema S SS Lee Nara N

Cell reports 20200601 13

Selective assembly of influenza virus segments into virions is proposed to be mediated through intersegmental RNA-RNA interactions. Here, we developed a method called 2CIMPL that includes proximity ligation under native conditions to identify genome-wide RNA duplexes. Interactions between all eight segments were observed at multiple sites along a given segment and are concentrated at hotspots. Furthermore, synonymous nucleotide changes in a hotspot decreased the formation of RNA-RNA interactions ...[more]

PMID: 32610124

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Project description: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.

Project description:The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

Dataset Information

Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network.

Publications

Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network.

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