Interaction of Host Nucleolin with Influenza A Virus Nucleoprotein in the Early Phase of Infection Limits the Late Viral Gene Expression

AbstractThe M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.Author summaryInfluenza A virus (IAV) pandemics arise when a virus adapted to a non-human host overcomes species barriers to successfully infect humans and sustain human-to-human transmission. To gauge the adaptive potential and therefore pandemic risk posed by a particular IAV, it is critical to understand the mechanisms underlying viral adaptation to human hosts. Here, we focused on the role of one of IAV’s eight gene segments, the M segment, in host adaptation. Comparing the growth of IAVs with avian- and human-derived M segments in avian and mammalian systems revealed that the avian M segment restricts viral growth specifically in mammalian cells. We show that the mechanism underlying this host range phenotype is a dysregulation of viral gene expression when the avian IAV M segment is transcribed in mammalian cells. In particular, excess production of the M2 protein results in viral interference with cellular functions on which the virus relies. Our results therefore reveal that the use of cellular machinery to control viral gene expression leaves the virus vulnerable to over- or under-production of critical viral gene products in a new host species.

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Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

Journal of General Virology ◽

10.1099/0022-1317-72-11-2661 ◽

1991 ◽

Vol 72 (11) ◽

pp. 2661-2670 ◽

Cited By ~ 7

Author(s):

J. Mukaigawa ◽

E. Hatada ◽

R. Fukuda ◽

K. Shimizu

Keyword(s):

Gene Expression ◽

Influenza A Virus ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene

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Downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza A virus vaccines

Future Virology ◽

10.2217/fvl.15.31 ◽

2015 ◽

Vol 10 (6) ◽

pp. 715-730 ◽

Cited By ~ 18

Author(s):

Steven F Baker ◽

Aitor Nogales ◽

Luis Martínez-Sobrido

Keyword(s):

Gene Expression ◽

Codon Usage ◽

Influenza A Virus ◽

Codon Usage Bias ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene ◽

Novel Influenza A

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Analysis of influenza a vtrus mutants having defects in the control of viral gene expression

Virus Research ◽

10.1016/0168-1702(88)90270-5 ◽

1988 ◽

Vol 11 ◽

pp. 92

Author(s):

R. Fukuda ◽

E. Hatada ◽

J. Mukaigawa ◽

K. Shimizu ◽

A. Ishihama

Keyword(s):

Gene Expression ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene

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An alternative AUG codon in segment 5 of the 2009 pandemic influenza A virus is a swine-derived virulence motif

10.1101/738427 ◽

2019 ◽

Cited By ~ 1

Author(s):

Helen M. Wise ◽

Eleanor Gaunt ◽

Jihui Ping ◽

Barbara Holzer ◽

Seema Jasim ◽

...

Keyword(s):

Influenza A Virus ◽

Untranslated Region ◽

Influenza A ◽

H1n1 Virus ◽

Viral Gene Expression ◽

Viral Gene ◽

Genetic Features ◽

Np Gene

AbstractThe 2009 influenza A virus (IAV) pandemic (pdm2009) was caused by a swine H1N1 virus with several atypical genetic features. Here, we investigate the origin and significance of an upstream AUG (uAUG) codon in the 5’-untranslated region of the NP gene. Phylogeny indicated that the uAUG codon arose in the classical swine IAV lineage in the mid 20th Century, and has become fixed in the current triple reassortant, variant pdm2009 swine IAV and human pdm2009 lineages. Functionally, it supports leaky ribosomal initiation in vitro and in vivo to produce two isoforms of NP: canonical, and a longer “eNP”. The uAUG codon had little effect on viral gene expression or replication in vitro. However, in both murine and porcine models of IAV infection, removing the uAUG codon gene attenuated pdm2009 virus pathogenicity. Thus, the NP uAUG codon is a virulence factor for swine IAVs with proven zoonotic ability.

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Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA

Journal of Virology ◽

10.1128/jvi.00528-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 15

Author(s):

Carina F. Pereira ◽

Eliot K. C. Read ◽

Helen M. Wise ◽

Maria J. Amorim ◽

Paul Digard

Keyword(s):

Influenza A Virus ◽

Nuclear Export ◽

Influenza A ◽

Matrix Protein ◽

Rna Binding ◽

Viral Gene Expression ◽

Mutant Virus ◽

Viral Gene ◽

Ns1 Protein ◽

Viral Mrnas

ABSTRACT Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.

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Influenza Virus NS1 Protein-RNA Interactome Reveals Intron Targeting

Journal of Virology ◽

10.1128/jvi.01634-18 ◽

2018 ◽

Vol 92 (24) ◽

Cited By ~ 10

Author(s):

Liang Zhang ◽

Juan Wang ◽

Raquel Muñoz-Moreno ◽

Min Kim ◽

Ramanavelan Sakthivel ◽

...

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Rna Binding ◽

Influenza Virus Infection ◽

Viral Gene Expression ◽

Viral Gene ◽

Ns1 Protein ◽

Cellular Processes

ABSTRACTThe 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.IMPORTANCEA 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.

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Pleotropic effects of mutations in the effector domain of influenza A virus NS1 protein

10.1101/390690 ◽

2018 ◽

Author(s):

Carina F. Pereira ◽

Helen M. Wise ◽

Dominic Kurian ◽

Rute M. Pinto ◽

Maria J. Amorim ◽

...

Keyword(s):

Influenza A Virus ◽

Nuclear Export ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene ◽

Ns1 Protein ◽

Innate Immune Responses ◽

Viral Mrnas ◽

Effector Domain ◽

Interaction Partners

AbstractObjectiveThe multifunctional NS1 protein of influenza A virus has roles in antagonising cellular innate immune responses and promoting viral gene expression. To better understand the interplay between these functions, we tested the effects of NS1 effector domain mutations known to affect homo-dimerisation or interactions with cellular PI3 kinase or Trim25 on NS1 ability to promote nuclear export of viral mRNAs.ResultsThe NS1 dimerisation mutant W187R retained the functions of binding cellular NXF1 as well as stabilising NXF1 interaction with viral segment 7 mRNAs and promoting their nuclear export. Two PI3K-binding mutants, NS1 Y89F and Y89A still bound NXF1 but no longer promoted NXF1 interactions with segment 7 mRNA or its nuclear export. The Trim25-binding mutant NS1 E96A/E97A bound NXF1 and supported NXF1 interactions with segment 7 mRNA but no longer supported mRNA nuclear export. Analysis of WT and mutant NS1 interaction partners identified hsp70 as specifically binding to NS1 E96A/E97A. Whilst these data suggest the possibility of functional links between NS1’s effects on intracellular signalling and its role in viral mRNA nuclear export, they also indicate potential pleiotropic effects of the NS1 mutations; in the case of E96A/E97A possibly via disrupted protein folding leading to chaperone recruitment.

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Infection- and procedure-dependent effects on pulmonary gene expression in the early phase of influenza A virus infection in mice

BMC Microbiology ◽

10.1186/1471-2180-13-293 ◽

2013 ◽

Vol 13 (1) ◽

pp. 293 ◽

Cited By ~ 19

Author(s):

Matthias Preusse ◽

Mohamed A Tantawy ◽

Frank Klawonn ◽

Klaus Schughart ◽

Frank Pessler

Keyword(s):

Gene Expression ◽

Virus Infection ◽

Influenza A Virus ◽

Early Phase ◽

Influenza A ◽

Influenza A Virus Infection

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A common pattern of influenza A virus single cell gene expression heterogeneity governs the innate antiviral response to infection

10.1101/858373 ◽

2019 ◽

Cited By ~ 2

Author(s):

J. Cristobal Vera ◽

Jiayi Sun ◽

Yen Ting Lin ◽

Jenny Drnevich ◽

Ruian Ke ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Host Cell ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene ◽

Cell Level ◽

Common Pattern ◽

Gene Expression Heterogeneity ◽

Infected Cells

ABSTRACTViral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual, single virion-infected cells. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. Our analyses reveal that viral NS segment gene expression diverges from that of the rest of the viral genome within a subset of infected cells, and that this unique pattern of NS segment expression can play a dominant role in shaping the host cell response to infection. Finally, we show that seasonal human H1N1 and H3N2 strains differ significantly in patterns of host anti-viral gene transcriptional heterogeneity at the single cell level. Altogether, these data reveal a common pattern of viral gene expression heterogeneity across human IAV subtypes that can serve as a major determinant of antiviral gene activation.

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