scholarly journals Influenza Virus NS1 Protein Counteracts PKR-Mediated Inhibition of Replication

2000 ◽  
Vol 74 (13) ◽  
pp. 6203-6206 ◽  
Author(s):  
Michael Bergmann ◽  
Adolfo Garcia-Sastre ◽  
Elena Carnero ◽  
Hubert Pehamberger ◽  
Klaus Wolff ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Protein Kinase ◽  
Gene Knockout ◽  
Ns1 Protein ◽  
Double Stranded Rna ◽  
Major Function ◽  
Ns1 Gene ◽  
Biological Relationship ◽  
Knockout Virus

ABSTRACT The availability of an influenza virus NS1 gene knockout virus (delNS1 virus) allowed us to establish the significance of the biological relationship between the influenza virus NS1 protein and double-stranded-RNA-activated protein kinase (PKR) in the life cycle and pathogenicity of influenza virus. Our results show that the lack of functional PKR permits the delNS1 virus to replicate in otherwise nonpermissive hosts, suggesting that the major function of the influenza virus NS1 protein is to counteract or prevent the PKR-mediated antiviral response.

scholarly journals A role for autophagolysosomes in dengue virus 3 production in HepG2 cells

2009 ◽  
Vol 90 (5) ◽  
pp. 1093-1103 ◽  
Author(s):  
Atefeh Khakpoor ◽  
Mingkwan Panyasrivanit ◽  
Nitwara Wikan ◽  
Duncan R. Smith
Keyword(s):  
Life Cycle ◽  
Confocal Microscopy ◽  
Dengue Virus ◽  
Hepg2 Cells ◽  
Cathepsin D ◽  
Fusion Inhibitor ◽  
Evidence Based ◽  
Ns1 Protein ◽  
Double Stranded Rna ◽  
A Site

We have recently proposed that amphisomes act as a site for translation and replication of dengue virus (DENV)-2 and that DENV-2 entry and replication are linked through an ongoing association with membranes of an endosomal–autophagosomal lineage. In this report, we present the results of an investigation into the interaction between DENV-3 and the autophagy machinery. Critically, treatment with the lysosomal fusion inhibitor l-asparagine differentiated the interaction of DENV-3 from that of DENV-2. Inhibition of fusion of autophagosomes and amphisomes with lysosomes resulted in decreased DENV-3 production, implying a role for the autophagolysosome in the DENV-3 life cycle. Evidence based upon the co-localization of LC3 and cathepsin D with double stranded RNA and NS1 protein, as assessed by confocal microscopy, support a model in which DENV-3 interacts with both amphisomes and autophagolysosomes. These results demonstrate that the interactions between DENV and the host cell autophagy machinery are complex and may be determined in part by virus-encoded factors.

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

2016 ◽  
Vol 90 (7) ◽  
pp. 3661-3675 ◽  
Author(s):  
Sathya N. Thulasi Raman ◽  
Guanqun Liu ◽  
Hyun Mi Pyo ◽  
Ya Cheng Cui ◽  
Fang Xu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Virus Infection ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Ns1 Protein ◽  
Antiviral Protein ◽  
Virus Life Cycle ◽  
Interaction Partners ◽  
Infected Cells

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.

scholarly journals A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Benjamin R. King ◽  
Dylan Hershkowitz ◽  
Philip L. Eisenhauer ◽  
Marion E. Weir ◽  
Christopher M. Ziegler ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Antiviral Activity ◽  
Viral Life Cycle ◽  
Host Protein ◽  
Data Set ◽  
Double Stranded Rna ◽  
Junin Virus ◽  
Protein Interactome ◽  
Junín Virus

ABSTRACT Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV. IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

scholarly journals Amelioration of Influenza Virus Pathogenesis in Chickens Attributed to the Enhanced Interferon-Inducing Capacity of a Virus with a Truncated NS1 Gene

2006 ◽  
Vol 81 (4) ◽  
pp. 1838-1847 ◽  
Author(s):  
Angela N. Cauthen ◽  
David E. Swayne ◽  
Margaret J. Sekellick ◽  
Philip I. Marcus ◽  
David L. Suarez
Keyword(s):  
Influenza Virus ◽  
High Titer ◽  
Kidney Tissue ◽  
Ns1 Protein ◽  
Phenotypic Differences ◽  
Embryo Cells ◽  
Ns1 Gene ◽  
Virus Strains ◽  
Lethal Doses

ABSTRACT Avian influenza virus (AIV) A/turkey/Oregon/71-SEPRL (TK/OR/71-SEPRL) (H7N3) encodes a full-length NS1 protein and is a weak inducer of interferon (IFN). A variant, TK/OR/71-delNS1 (H7N3), produces a truncated NS1 protein and is a strong inducer of IFN. These otherwise genetically related variants differ 20-fold in their capacities to induce IFN in primary chicken embryo cells but are similar in their sensitivities to the action of IFN. Furthermore, the weak IFN-inducing strain actively suppresses IFN induction in cells that are otherwise programmed to produce it. These phenotypic differences are attributed to the enhanced IFN-inducing capacity that characterizes type A influenza virus strains that produce defective NS1 protein. The pathogenesis of these two variants was evaluated in 1-day-old and 4-week-old chickens. The cell tropisms of both viruses were similar. However, the lesions in chickens produced by the weak IFN inducer were more severe and differed somewhat in character from those observed for the strong IFN inducer. Differences in lesions included the nature of inflammation, the rate of resolution of the infection, and the extent of viral replication and/or virus dissemination. The amelioration of pathogenesis is attributed to the higher levels of IFN produced by the variant encoding the truncated NS1 protein and the antiviral state subsequently induced by that IFN. The high titer of virus observed in kidney tissue (≈109 50% embryo lethal doses/g) from 1-day-old chickens infected intravenously by the weak IFN-inducing strain is attributed to the capacity of chicken kidney cells to activate the hemagglutinin fusion peptide along with their unresponsiveness to inducers of IFN as measured in vitro. Thus, the IFN-inducing capacity of AIV appears to be a significant factor in regulating the pathogenesis, virulence, and viral transmission of AIV in chickens. This suggests that the IFN-inducing and IFN induction suppression phenotypes of AIV should be considered when characterizing strains of influenza virus.

scholarly journals Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

1991 ◽  
Vol 11 (2) ◽  
pp. 1092-1098 ◽  
Author(s):  
F V Alonso-Caplen ◽  
R M Krug
Keyword(s):  
Influenza Virus ◽  
Adenovirus Vector ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Fraction ◽  
Ns1 Protein ◽  
Cis Acting ◽  
Ns1 Gene ◽  
Infected Cells ◽  
Adenovirus Recombinant

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.

scholarly journals The NS1 Protein of the 1918 Pandemic Influenza Virus Blocks Host Interferon and Lipid Metabolism Pathways

2009 ◽  
Vol 83 (20) ◽  
pp. 10557-10570 ◽  
Author(s):  
Rosalind Billharz ◽  
Hui Zeng ◽  
Sean C. Proll ◽  
Marcus J. Korth ◽  
Sharon Lederer ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Lipid Metabolism ◽  
Host Response ◽  
Infection Model ◽  
Type I ◽  
Ns1 Protein ◽  
Cell Infection ◽  
Human Influenza Virus ◽  
Proinflammatory Mediators ◽  
Ns1 Gene

ABSTRACT The “Spanish influenza” of 1918 claimed an unprecedented number of lives, yet the determinants of virulence for this virus are still not fully understood. Here, we used functional genomics and an in vitro human lung epithelial cell infection model to define the global host transcriptional response to the eight-gene 1918 virus. To better understand the role of the 1918 virus NS1 gene, we also evaluated the host response to a reassortant 1918 virus containing the NS1 gene from A/Texas/36/91 (a seasonal isolate of human influenza virus), as well as the host response to a reassortant of A/Texas/36/91 containing the 1918 NS1 gene. Genomic analyses revealed that the 1918 virus blocked the transcription of multiple interferon-stimulated genes and also downregulated a network of genes associated with lipid metabolism. In contrast, the expression of genes encoding chemokines and cytokines, which serve to attract infiltrating immune cells, was upregulated. Viruses containing the NS1 gene from A/Texas/36/91 induced a significant increase in type I interferon signaling but did not repress lipid metabolism. The 1918 NS1 gene may therefore have contributed to the virulence of the 1918 pandemic virus by disrupting the innate immune response, inducing hypercytokinemia, and by blocking the transcription of certain lipid-based proinflammatory mediators that function as part of the host antiviral response.

Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA

1991 ◽  
Vol 11 (2) ◽  
pp. 1092-1098
Author(s):  
F V Alonso-Caplen ◽  
R M Krug
Keyword(s):  
Influenza Virus ◽  
Adenovirus Vector ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Fraction ◽  
Ns1 Protein ◽  
Cis Acting ◽  
Ns1 Gene ◽  
Infected Cells ◽  
Adenovirus Recombinant

Influenza virus NS1 mRNA is spliced by host nuclear enzymes to form NS2 mRNA, and this splicing is regulated in infected cells such that the steady-state amount of spliced NS2 mRNA is only about 10% of that of unspliced NS1 mRNA. This regulation would be expected to result from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus. To determine whether the rate of splicing of NS1 mRNA was controlled by trans factors in influenza virus-infected cells, the NS1 gene was inserted into an adenovirus vector. The rates of splicing of NS1 mRNA in cells infected with this vector and in influenza virus-infected cells were measured by pulse-labeling with [3H]uridine. The rates of splicing of NS1 mRNA in the two systems were not significantly different, strongly suggesting that the rate of splicing of NS1 mRNA in influenza virus-infected cells is controlled solely by cis-acting sequences in NS1 mRNA itself. In contrast to the rate of splicing, the extent of splicing of NS1 mRNA in the cells infected by the adenovirus recombinant was dramatically increased relative to that occurring in influenza virus-infected cells. This could be attributed largely, if not totally, to a block in the nucleocytoplasmic transport of unspliced NS1 mRNA in the recombinant-infected cells. Most of the unspliced NS1 mRNA was in the nuclear fraction, and no detectable NS1 protein was synthesized. When the 3' splice site of NS1 mRNA was inactivated by mutation, NS1 mRNA was transported and translated, indicating that the transport block occurred because NS1 rRNA was committed to the splicing pathway. This transport block is apparently obviated in influenza virus-infected cells. These experiments demonstrate the important role of the nucleocytoplasmic transport of unspliced NS1 mRNA in regulating the extent of splicing of NS1 mRNA.

scholarly journals West Nile Virus-Induced Interferon Production Is Mediated by the Double-Stranded RNA-Dependent Protein Kinase PKR

2007 ◽  
Vol 81 (20) ◽  
pp. 11148-11158 ◽  
Author(s):  
Felicia D. Gilfoy ◽  
Peter W. Mason
Keyword(s):  
West Nile Virus ◽  
Protein Kinase ◽  
Gene Knockout ◽  
Poly I:C ◽  
Type I ◽  
West Nile ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Dependent Protein ◽  
Chemical Inhibition

ABSTRACT Cells carry a variety of molecules, referred to as pathogen recognition receptors (PRRs), which are able to sense invading pathogens. Interaction of PRRs with viral compounds instigates a signaling pathway(s), resulting in the activation of genes, including those for type I interferon (IFN), which are critical for an effective antiviral response. Here we demonstrate that the double-stranded RNA (dsRNA)-dependent protein kinase PKR, which has been shown to function as a PRR in cells treated with the dsRNA mimetic poly(I:C), serves as a PRR in West Nile virus (WNV)-infected cells. Evidence for PKR's role as a PRR was obtained from both human and murine cells. Using mouse embryonic fibroblasts (MEFs), we demonstrated that PKR gene knockout, posttranscriptional gene silencing of PKR mRNA using small interfering RNA (siRNA), and chemical inhibition of PKR function all interfered with IFN synthesis following WNV infection. In three different human cell lines, siRNA knockdown and chemical inhibition of PKR blocked WNV-induced IFN synthesis. Using the same approaches, we demonstrated that PKR was not necessary for Sendai virus-induced IFN synthesis, suggesting that PKR is particularly important for recognition of WNV infection. Taken together, our data suggest that PKR could serve as a PRR for recognition of WNV infection.

scholarly journals Clonogenic Assay of Type A Influenza Viruses Reveals Noninfectious Cell-Killing (Apoptosis-Inducing) Particles

2008 ◽  
Vol 82 (6) ◽  
pp. 2673-2680 ◽  
Author(s):  
John M. Ngunjiri ◽  
Margaret J. Sekellick ◽  
Philip I. Marcus
Keyword(s):  
Influenza Virus ◽  
Vero Cells ◽  
Cell Killing ◽  
Influenza Viruses ◽  
Ns1 Protein ◽  
Full Size ◽  
Rate Limiting Step ◽  
A Cell ◽  
Ns1 Gene ◽  
Colony Forming Capacity

ABSTRACT Clonogenic (single-cell plating) assays were used to define and quantify subpopulations of two genetically closely related variants of influenza virus A/TK/OR/71 that differed primarily in the size of the NS1 gene product; they expressed a full-size (amino acids [aa] 1 to 230) or truncated (aa 1 to 124) NS1 protein. Monolayers of Vero cells were infected with different amounts of virus, monodispersed, and plated. Cell survival curves were generated from the fraction of cells that produced visible colonies as a function of virus multiplicity. The exponential loss of colony-forming capacity at low multiplicities demonstrated that a single virus particle sufficed to kill a cell. The ratios of cell-killing particles (CKP) to plaque-forming particles (PFP) were 1:1 and 7:1 in populations of variants NS11-124 and NS11-230, respectively. This study revealed a new class of particles in influenza virus populations—noninfectious CKP. Both infectious and noninfectious CKP were 6.3 times more resistant to UV radiation than PFP activity. Based on UV target theory, a functional polymerase subunit was implicated in a rate-limiting step in cell killing. Since influenza viruses kill cells by apoptosis (programmed cell death), CKP are functionally apoptosis-inducing particles. Noninfectious CKP are present in excess of PFP in virus populations with full-size NS1 and induce apoptosis that is temporally delayed and morphologically different than that initiated by infectious CKP present in the virus population expressing truncated NS1. The identification and quantification of both infectious and noninfectious CKP defines new phenotypes in influenza virus populations and presents a challenge to determine their role in regulating infectivity, pathogenesis, and vaccine efficacy.

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