DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

ABSTRACTDDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation.IMPORTANCEDDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.

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Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication

Journal of Virology ◽

10.1128/jvi.00504-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7603-7612 ◽

Cited By ~ 30

Author(s):

Susana de Lucas ◽

Joan Peredo ◽

Rosa María Marión ◽

Carmen Sánchez ◽

Juan Ortín

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Particle Production ◽

Nonstructural Protein ◽

A549 Cells ◽

Influenza Virus Infection ◽

Ns1 Protein ◽

Infected Cells

ABSTRACT The influenza A virus genome consists of 8 negative-stranded RNA segments. NS1 is a nonstructural protein that participates in different steps of the virus infectious cycle, including transcription, replication, and morphogenesis, and acts as a virulence factor. Human Staufen1 (hStau1), a protein involved in the transport and regulated translation of cellular mRNAs, was previously identified as a NS1-interacting factor. To investigate the possible role of hStau1 in the influenza virus infection, we characterized the composition of hStau1-containing granules isolated from virus-infected cells. Viral NS1 protein and ribonucleoproteins (RNPs) were identified in these complexes by Western blotting, and viral mRNAs and viral RNAs (vRNAs) were detected by reverse transcription (RT)-PCR. Also, colocalization of hStau1 with NS1, nucleoprotein (NP), and PA in the cytosol of virus-infected cells was shown by immunofluorescence. To analyze the role of hStau1 in the infection, we downregulated its expression by gene silencing. Human HEK293T cells or A549 cells were silenced using either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting four independent sites in the hStau1 mRNA. The yield of influenza virus was reduced 5 to 10 times in the various hStau1-silenced cells compared to that in control silenced cells. The expression levels of viral proteins and their nucleocytoplasmic localization were not affected upon hStau1 silencing, but virus particle production, as determined by purification of virions from supernatants, was reduced. These results indicate a role for hStau1 in late events of the influenza virus infection, possibly during virus morphogenesis.

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Influenza Virus Evades Innate and Adaptive Immunity via the NS1 Protein

Journal of Virology ◽

10.1128/jvi.02381-05 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6295-6304 ◽

Cited By ~ 206

Author(s):

Ana Fernandez-Sesma ◽

Svetlana Marukian ◽

Barbara J. Ebersole ◽

Dorothy Kaminski ◽

Man-Seong Park ◽

...

Keyword(s):

Innate Immunity ◽

Influenza Virus ◽

Virus Infection ◽

Adaptive Immunity ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Virus Infection ◽

Type I ◽

Ns1 Protein ◽

Dc Maturation

ABSTRACT Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1β, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-α/β, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.

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Interferon-β Stimulation Elicited by the Influenza Virus Is Regulated by the Histone Methylase Dot1L through the RIG-I-TRIM25 Signaling Axis

Cells ◽

10.3390/cells9030732 ◽

2020 ◽

Vol 9 (3) ◽

pp. 732 ◽

Cited By ~ 1

Author(s):

Laura Marcos-Villar ◽

Estanislao Nistal-Villan ◽

Noelia Zamarreño ◽

Urtzi Garaigorta ◽

Pablo Gastaminza ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Nuclear Translocation ◽

Control Point ◽

Influenza Virus Infection ◽

Nuclear Factor Κb ◽

Antiviral Response ◽

General Role ◽

Infected Cells

Influenza virus infection increases the methylation of lysine 79 of histone 3 catalyzed by the Dot1L enzyme. The role of Dot1L against infections was highlighted by an increase of influenza A and vesicular stomatitis virus replication in Dot1L-inhibited cells mediated by a decreased antiviral response. Interferon-beta (IFN-β) reporter assays indicate that Dot1L is involved in the control of retinoic acid-inducible gene-I protein (RIG-I) signaling. Accordingly, Dot1L inhibition decreases the IFN-β promoter stimulation and RIG-I- mitochondria-associated viral sensor (RIG-I-MAVS) association upon viral infection. Replication of an influenza A virus lacking NS1 (delNS1), incapable of counteracting the antiviral response, is not affected by Dot1L inhibition. Consequently, RIG-I-MAVS association and nuclear factor–κB (NF-κB) nuclear translocation, are not affected by the Dot1L inhibition in delNS1 infected cells. Restoration of NS1 expression in trans also reinstated Dot1L as a regulator of the RIG-I-dependent signaling in delNS1 infections. Interferon-inducible E3 ligase tripartite motif-containing protein 25 (TRIM25) expression increases in influenza virus infected cells, but Dot1L inhibition reduces both the TRIM25 expression and TRIM25 protein levels. TRIM25 overexpression reverses the defective innate response mediated by Dot1L inhibition elicited upon virus infection or by overexpression of RIG-I signaling intermediates. Thus, TRIM25 is a control point of the RIG-I recognition pathway controlled by Dot1L and may have a general role in RNA viruses recognized by the RIG-I sensor.

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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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Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

mBio ◽

10.1128/mbio.03315-21 ◽

2022 ◽

Author(s):

Sho Miyamoto ◽

Masahiro Nakano ◽

Takeshi Morikawa ◽

Ai Hirabayashi ◽

Ryoma Tamura ◽

...

Keyword(s):

Influenza Virus ◽

Life Cycle ◽

Viral Genome ◽

Influenza A ◽

Genome Replication ◽

Ribonucleoprotein Complex ◽

Virus Life Cycle ◽

Viral Genome Replication ◽

Infected Cells ◽

Nuclear Domains

Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process.

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Impact of influenza virus during pregnancy: from disease severity to vaccine efficacy

Future Virology ◽

10.2217/fvl-2020-0024 ◽

2020 ◽

Vol 15 (7) ◽

pp. 441-453

Author(s):

Ana Vazquez-Pagan ◽

Rebekah Honce ◽

Stacey Schultz-Cherry

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Immune Responses ◽

Pregnant Women ◽

Disease Severity ◽

Influenza A ◽

Antiviral Treatment ◽

Influenza Virus Infection ◽

Severe Influenza ◽

The Impact

Pregnant women are among the individuals at the highest risk for severe influenza virus infection. Infection of the mother during pregnancy increases the probability of adverse fetal outcomes such as small for gestational age, preterm birth and fetal death. Animal models of syngeneic and allogeneic mating can recapitulate the increased disease severity observed in pregnant women and are used to define the mechanism(s) of that increased severity. This review focuses on influenza A virus pathogenesis, the unique immunological landscape during pregnancy, the impact of maternal influenza virus infection on the fetus and the immune responses at the maternal–fetal interface. Finally, we summarize the importance of immunization and antiviral treatment in this population and highlight issues that warrant further investigation.

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A Fungal Cu/Zn-Containing Superoxide Dismutase Enhances the Therapeutic Efficacy of a Plant Polyphenol Extract in Experimental Influenza Virus Infection

Zeitschrift für Naturforschung C ◽

10.1515/znc-2010-5-616 ◽

2010 ◽

Vol 65 (5-6) ◽

pp. 419-428 ◽

Cited By ~ 1

Author(s):

Julia Serkedjieva ◽

Tsvetanka Stefanova ◽

Ekaterina Krumova

Keyword(s):

Superoxide Dismutase ◽

Influenza Virus ◽

Virus Infection ◽

Protective Effect ◽

Influenza A ◽

Influenza Virus Infection ◽

Protective Role ◽

Combined Application ◽

Combined Use ◽

Experimental Influenza

The combined protective effect of a polyphenol-rich extract, isolated from Geranium sanguineum L. (PC), and a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced from the fungal strain Humicula lutea 103 (HL-SOD), in the experimental influenza A virus infection (EIVI) in mice, induced with the virus A/Aichi/2/68 (H3N2), was investigated. The combined application of HL-SOD and PC in doses, which by themselves do not defend significantly mice in EIVI, resulted in a synergistically increased protection, determined on the basis of protective indices and amelioration of lung injury. Lung weights and consolidation as well as infectious lung virus titers were all decreased significantly parallel to the reduction of the mortality rates; lung indices were raised. The excessive production of reactive oxygen species (ROS) by alveolar macrophages (aMØ) as well as the elevated levels of the lung antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), induced by EIVI, were brought to normal. For comparative reasons the combined protective effect of PC and vitamin C was investigated. The obtained results support the combined use of antioxidants for the treatment of influenza virus infection and in general indicate the beneficial protective role of combinations of viral inhibitors of natural origin with diverse modes of action.

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Severe Influenza Virus Infection in Children Admitted to the PICU: Comparison of Influenza A and Influenza B Virus Infection

Journal of Medical Virology ◽

10.1002/jmv.27400 ◽

2021 ◽

Author(s):

Pınar YAZICI ÖZKAYA ◽

Eşe Eda TURANLI ◽

Hamdi METİN ◽

Ayça Aydın UYSAL ◽

Candan ÇİÇEK ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza B Virus ◽

Influenza B ◽

Severe Influenza ◽

B Virus

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IFITM3 protects the heart during influenza virus infection

10.1101/518548 ◽

2019 ◽

Author(s):

Adam D. Kenney ◽

Temet M. McMichael ◽

Alexander Imas ◽

Nicholas M. Chesarino ◽

Lizhi Zhang ◽

...

Keyword(s):

Heart Rate ◽

Influenza Virus ◽

Virus Infection ◽

Cardiac Dysfunction ◽

Influenza A ◽

Transmembrane Protein ◽

Influenza Virus Infection ◽

Small Animal ◽

Heart Tissue ◽

Underlying Mechanisms

AbstractInfluenza virus primarily targets the lungs, but dissemination and damage to heart tissue is also known to occur in severe infections. Despite this knowledge, influenza virus-induced cardiac pathogenesis and its underlying mechanisms have been difficult to study due to a lack of small animal models. In humans, polymorphisms in the gene encoding interferon-induced transmembrane protein 3 (IFITM3), an antiviral restriction factor, are associated with susceptibility to severe influenza, but whether IFITM3 deficiencies contribute to other aspects of pathogenesis, including cardiac dysfunction, is unknown. We now show that IFITM3 deficiency in a newly generated knockout (KO) mouse model exacerbates illness and mortality following influenza A virus infection. Enhanced pathogenesis correlated with increased replication of virus in the lungs, spleens, and hearts of KO mice relative to wildtype (WT) mice. IFITM3 KO mice exhibited normal cardiac function at baseline, but developed severely aberrant electrical activity upon infection, including decreased heart rate and irregular, arrhythmic RR (interbeat) intervals. In contrast, WT mice exhibited a mild decrease in heart rate without irregularity of RR intervals. Heightened cardiac virus titers and electrical dysfunction in KO animals was accompanied by increased activation of fibrotic pathways and fibrotic lesions in the heart. Our findings reveal an essential role for IFITM3 in controlling influenza virus replication and pathogenesis in heart tissue and establish IFITM3 KO mice as a powerful model to study virus-induced cardiac dysfunction.

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Routine blood parameters are helpful for early identification of influenza infection in children

10.21203/rs.3.rs-54705/v2 ◽

2020 ◽

Author(s):

Ronghe Zhu ◽

Cuie Chen ◽

Qiu Wang ◽

Xixi Zhang ◽

Chaosheng Lu ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A Virus ◽

Early Identification ◽

Influenza A ◽

Influenza Infection ◽

Influenza Virus Infection ◽

Cutoff Value ◽

Routine Blood ◽

Influenza A Virus Infection

Abstract Purpose Routine blood parameters, such as the lymphocyte (LYM) count, platelet (PLT) count, lymphocyte-to-monocyte ratio (LMR), neutrophil-to-lymphocyte ratio (NLR), LYM*PLT and mean platelet volume-to-platelet ratio (MPV/PLT), are widely used to predict the prognosis of infectious diseases. We aimed to explore the value of these parameters in the early identification of influenza virus infection in children.Methods We conducted a single-center, retrospective, observational study of fever with influenza-like symptoms in pediatric outpatients from different age groups and evaluated the predictive value of various routine blood parameters measured within 48 hours of the onset of fever for influenza virus infection.Results The LYM count, PLT count, LMR and LYM*PLT were lower, and the NLR and MPV/PLT were higher in children with an influenza infection (PCR-confirmed and symptomatic). The LYM count, LMR and LYM*PLT in the influenza infection group were lower in the 1- to 6-year-old subgroup, and the LMR and LYM*PLT in the influenza infection group were lower in the >6-year-old subgroup. In the 1- to 6-year-old subgroup, the cutoff value of the LMR for predicting influenza A virus infection was 3.75, the sensitivity was 81.87%, the specificity was 84.31%, and the area under the curve (AUC) was 0.886; the cutoff value of the LMR for predicting influenza B virus infection was 3.71, the sensitivity was 73.58%, the specificity was 84.31%, and the AUC was 0.843. In the >6-year-old subgroup, the cutoff value of the LMR for predicting influenza A virus infection was 3.05, the sensitivity was 89.27%, the specificity was 89.61%, and the AUC was 0.949; the cutoff value of the LMR for predicting influenza B virus infection was 2.88, the sensitivity was 83.19%, the specificity was 92.21%, and the AUC was 0.924.Conclusions Routine blood tests are simple, inexpensive and easy to perform, and they are useful for the early identification of influenza virus infection in children. The LMR had the strongest predictive value for influenza virus infection in children older than 1 year, particularly influenza A virus infection.

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