scholarly journals RuvB-Like Protein 2 Interacts with the NS1 Protein of Influenza A Virus and Affects Apoptosis That Is Counterbalanced by Type I Interferons

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1038
Author(s):  
Yimeng Wang ◽  
Jianhong Zhou ◽  
Samuel G. Mackintosh ◽  
Yuchun Du
Keyword(s):  
Influenza A ◽  
A549 Cells ◽  
Vero Cells ◽  
Type I ◽  
Ns1 Protein ◽  
Wild Type ◽  
Protein Levels ◽  
Infected Cells ◽  
Resistance To Infection ◽  
Induced Apoptosis

The NS1 protein of influenza A virus (IAV) plays important roles in viral pathogenesis and host immune response. Through a proteomic approach, we have identified RuvB-like proteins 1 and 2 (RuvBL1 and RuvBL2) as interacting partners of the NS1 protein of IAVs. Infection of human lung A549 cells with A/PR/8/34 (PR8) virus resulted in reductions in the protein levels of RuvBL2 but not RuvBL1. Further studies with RuvBL2 demonstrated that the NS1-RuvBL2 interaction is RNA-independent, and RuvBL2 binds the RNA-binding domain of the NS1. Infection of interferon (IFN)-deficient Vero cells with wild-type or delNS1 PR8 virus reduced RuvBL2 protein levels and induced apoptosis; delNS1 virus caused more reductions in RuvBL2 protein levels and induced more apoptosis than did wild-type virus. Knockdown of RuvBL2 by siRNAs induced apoptosis and overexpression of RuvBL2 resulted in increased resistance to infection-induced apoptosis in Vero cells. These results suggest that a non-NS1 viral element or elements induce apoptosis by suppressing RuvBL2 protein levels, and the NS1 inhibits the non-NS1 viral element-induced apoptosis by maintaining RuvBL2 abundance in infected cells in the absence of IFN influence. In contrast to Vero cells, infection of IFN-competent A549 cells with PR8 virus caused reductions in RuvBL2 protein levels but did not induce apoptosis. Concomitantly, pretreatment of Vero cells with a recombinant IFN resulted in resistance to infection-induced apoptosis. These results demonstrate that the infection-induced, RuvBL2-regulated apoptosis in infected cells is counterbalanced by IFN survival signals. Our results reveal a novel mechanism underlying the infection-induced apoptosis that can be modulated by the NS1 and type I IFN signaling in IAV-infected cells.

scholarly journals Dicer is involved in protection against influenza A virus infection

2007 ◽  
Vol 88 (10) ◽  
pp. 2627-2635 ◽  
Author(s):  
Alexey A. Matskevich ◽  
Karin Moelling
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Virus Production ◽  
Vero Cells ◽  
Antiviral Response ◽  
Protein Levels ◽  
Infected Cells ◽  
Influenza A Virus Infection

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

scholarly journals NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

2002 ◽  
Vol 76 (4) ◽  
pp. 1617-1625 ◽  
Author(s):  
O. P. Zhirnov ◽  
T. E. Konakova ◽  
T. Wolff ◽  
H.-D. Klenk
Keyword(s):  
Influenza A ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Vero Cells ◽  
Ns1 Protein ◽  
Influenza A Viruses ◽  
Caspase Cleavage ◽  
Chicken Embryos ◽  
Ns1 Gene ◽  
Induced Apoptosis

ABSTRACT Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.

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

2010 ◽  
Vol 84 (15) ◽  
pp. 7603-7612 ◽  
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.

scholarly journals Properties of H7N7 influenza A virus strain SC35M lacking interferon antagonist NS1 in mice and chickens

2007 ◽  
Vol 88 (5) ◽  
pp. 1403-1409 ◽  
Author(s):  
Georg Kochs ◽  
Iris Koerner ◽  
Lena Thiel ◽  
Sonja Kothlow ◽  
Bernd Kaspers ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Virulence Factor ◽  
Influenza A ◽  
Severe Disease ◽  
A549 Cells ◽  
Type I ◽  
Upper Respiratory Tract ◽  
Structural Protein ◽  
Wild Type ◽  
Ns1 Gene

Non-structural protein NS1 of influenza A virus counteracts the host immune response by blocking the synthesis of type I interferon (IFN). As deletion of the complete NS1 gene has to date been reported only in the human H1N1 strain A/PR/8/34, it remained unclear whether NS1 is a non-essential virulence factor in other influenza A virus strains as well. In this report, the properties of NS1-deficient mutants derived from strain SC35M (H7N7) are described. A mutant of SC35M that completely lacks the NS1 gene was an excellent inducer of IFN in mammalian and avian cells in culture and, consequently, was able to multiply efficiently only in cell lines with defects in the type I IFN system. Virus mutants carrying C-terminally truncated versions of NS1 were less powerful inducers of IFN and were attenuated less strongly in human A549 cells. Although attenuated in wild-type mice, these mutants remained highly pathogenic for mice lacking the IFN-regulated antiviral factor Mx1. In contrast, the NS1-deficient SC35M mutant was completely non-pathogenic for wild-type mice, but remained pathogenic for mice lacking Mx1 and double-stranded RNA-activated protein kinase (PKR). Wild-type SC35M, but not the NS1-deficient mutant virus, was able to replicate in the upper respiratory tract of birds, but neither virus induced severe disease in adult chickens. Altogether, this study supports the view that NS1 represents a non-essential virulence factor of different influenza A viruses.

scholarly journals The Novel H7N9 Influenza A Virus NS1 Induces p53-Mediated Apoptosis of A549 Cells

2016 ◽  
Vol 38 (4) ◽  
pp. 1447-1458 ◽  
Author(s):  
Yinxia Yan ◽  
Yongming Du ◽  
Gefei Wang ◽  
Yuxue Deng ◽  
Rui Li ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Influenza A ◽  
Caspase 3 ◽  
Eastern China ◽  
A549 Cells ◽  
Epithelial Cell Line ◽  
Ns1 Protein ◽  
Protein Levels

Background: H7N9, emerged as an avian influenza virus outbreak in Eastern China in early 2013, and represented another major threat to global health. Roles of its NS1 protein, an essential viral factor, in regulating apoptosis remain unknown. Methods: Apoptotic effect and features of H7N9/NS1 in the human A549 alveolar basal epithelial cell line were examined by caspase 3/7 activity assay and western blotting of apoptotic associated proteins. Effects of H7N9/NS1on mitochondrial membrane potential were investigated by flow cytometry. Results: The expression of H7N9/NS1 in A549 cells activated caspase 3/7 and increased the protein levels of cleaved caspase 7 and cleaved poly (ADP-ribose) polymerase (PARP). H7N9/NS1-expressing A549 cells displayed a decrease in mitochondrial membrane potential. In addition, H7N9/NS1 increased the protein levels of total p53, p53 phosphorylated at Ser46 and Ser37, activated caspase 9, and the Bax/Bcl-2 ratio. Conclusion: Our results suggest that H7N9/NS1 protein causes the accumulation of p53 by increasing phosphorylation levels of p53 and the induction of mitochondrial dysfunction, which may contribute to H7N9/NS1-induced apoptosis in A549 cells.

scholarly journals Type I IFN Triggers RIG-I/TLR3/NLRP3-dependent Inflammasome Activation in Influenza A Virus Infected Cells

2013 ◽  
Vol 9 (4) ◽  
pp. e1003256 ◽  
Author(s):  
Julien Pothlichet ◽  
Isabelle Meunier ◽  
Beckley K. Davis ◽  
Jenny P-Y. Ting ◽  
Emil Skamene ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Inflammasome Activation ◽  
Type I ◽  
Type I Ifn ◽  
Infected Cells

Role of the chaperone protein 14-3-3ε in the regulation of influenza A virus-activated beta interferon

2021 ◽  
Author(s):  
Ee-Hong Tam ◽  
Yen-Chin Liu ◽  
Chian-Huey Woung ◽  
Helene Minyi Liu ◽  
Guan-Hong Wu ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Critical Role ◽  
Type I ◽  
Type I Ifn ◽  
Ns1 Protein ◽  
Chaperone Protein ◽  
Influenza A Virus Infection

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in RIG-I translocation to MAVS to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-β promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-β expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49 th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C-terminus-truncated NS1 with T49A mutation dramatically increases IFN-β mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-β expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in RIG-I translocation that induces type I IFN expression, and that NS1 protein prevents RIG-I translocation to mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-β suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

scholarly journals Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein

2004 ◽  
Vol 78 (8) ◽  
pp. 3880-3888 ◽  
Author(s):  
Ana M. Falcón ◽  
Rosa M. Marión ◽  
Thomas Zürcher ◽  
Paulino Gómez ◽  
Agustín Portela ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Ns1 Protein ◽  
Late Gene Expression ◽  
Infected Cells ◽  
Defective Rna ◽  
Nucleocytoplasmic Export

ABSTRACT Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.

scholarly journals The NS1 Protein of Influenza A Virus Blocks RIG-I-Mediated Activation of the Noncanonical NF-κB Pathway and p52/RelB-Dependent Gene Expression in Lung Epithelial Cells

2012 ◽  
Vol 86 (18) ◽  
pp. 10211-10217 ◽  
Author(s):  
Andrea Rückle ◽  
Emanuel Haasbach ◽  
Ilkka Julkunen ◽  
Oliver Planz ◽  
Christina Ehrhardt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Influenza A Virus ◽  
Influenza A ◽  
Target Gene ◽  
Lung Epithelial Cells ◽  
Ns1 Protein ◽  
Antiviral Immune Response ◽  
Lung Epithelial ◽  
Infected Cells

Influenza A virus (IAV) infection of epithelial cells activates NF-κB transcription factors via the canonical NF-κB signaling pathway, which modulates both the antiviral immune response and viral replication. Since almost nothing is known so far about a function of noncanonical NF-κB signaling after IAV infection, we tested infected cells for activation of p52 and RelB. We show that the viral NS1 protein strongly inhibits RIG-I-mediated noncanonical NF-κB activation and expression of the noncanonical target gene CCL19.

scholarly journals RIG-I Plays a Dominant Role in the Induction of Transcriptional Changes in Zika Virus-Infected Cells, which Protect from Virus-Induced Cell Death

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1476 ◽  
Author(s):  
Mirjam Schilling ◽  
Anne Bridgeman ◽  
Nicki Gray ◽  
Jonny Hertzog ◽  
Philip Hublitz ◽  
...  
Keyword(s):  
Cell Death ◽  
Zika Virus ◽  
Dominant Role ◽  
A549 Cells ◽  
Effective Control ◽  
Type I ◽  
Type I Ifn ◽  
Structural Protein ◽  
Induced Apoptosis ◽  
The Individual

The Zika virus (ZIKV) has received much attention due to an alarming increase in cases of neurological disorders including congenital Zika syndrome associated with infection. To date, there is no effective treatment available. An immediate response by the innate immune system is crucial for effective control of the virus. Using CRISPR/Cas9-mediated knockouts in A549 cells, we investigated the individual contributions of the RIG-I-like receptors MDA5 and RIG-I to ZIKV sensing and control of this virus by using a Brazilian ZIKV strain. We show that RIG-I is the main sensor for ZIKV in A549 cells. Surprisingly, we observed that loss of RIG-I and consecutive type I interferon (IFN) production led to virus-induced apoptosis. ZIKV non-structural protein NS5 was reported to interfere with type I IFN receptor signaling. Additionally, we show that ZIKV NS5 inhibits type I IFN induction. Overall, our study highlights the importance of RIG-I-dependent ZIKV sensing for the prevention of virus-induced cell death and shows that NS5 inhibits the production of type I IFN.

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