scholarly journals Pestivirus NproDirectly Interacts with Interferon Regulatory Factor 3 Monomer and Dimer

2016 ◽  
Vol 90 (17) ◽  
pp. 7740-7747 ◽  
Author(s):  
Keerthi Gottipati ◽  
Luis Marcelo F. Holthauzen ◽  
Nicolas Ruggli ◽  
Kyung H. Choi
Keyword(s):  
Viral Infection ◽  
Binding Site ◽  
Classical Swine Fever Virus ◽  
Active Form ◽  
Classical Swine Fever ◽  
Proteasomal Degradation ◽  
Interferon Regulatory Factor ◽  
Creb Binding Protein ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3

ABSTRACTInterferon regulatory factor 3 (IRF3) is a transcription factor involved in the activation of type I alpha/beta interferon (IFN-α/β) in response to viral infection. Upon viral infection, the IRF3 monomer is activated into a phosphorylated dimer, which induces the transcription of interferon genes in the nucleus. Viruses have evolved several ways to target IRF3 in order to subvert the innate immune response. Pestiviruses, such as classical swine fever virus (CSFV), target IRF3 for ubiquitination and subsequent proteasomal degradation. This is mediated by the viral protein Nprothat interacts with IRF3, but the molecular details for this interaction are largely unknown. We used recombinant Nproand IRF3 proteins and show that Nprointeracts with IRF3 directly without additional proteins and forms a soluble 1:1 complex. The full-length IRF3 but not merely either of the individual domains is required for this interaction. The interaction between Nproand IRF3 is not dependent on the activation state of IRF3, since Nprobinds to a constitutively active form of IRF3 in the presence of its transcriptional coactivator, CREB-binding protein (CBP). The results indicate that the Npro-binding site on IRF3 encompasses a region that is unperturbed by the phosphorylation and subsequent activation of IRF3 and thus excludes the dimer interface and CBP-binding site.IMPORTANCEThe pestivirus N-terminal protease, Npro, is essential for evading the host's immune system by facilitating the degradation of interferon regulatory factor 3 (IRF3). However, the nature of the Nprointeraction with IRF3, including the IRF3 species (inactive monomer versus activated dimer) that Nprotargets for degradation, is largely unknown. We show that classical swine fever virus Nproand porcine IRF3 directly interact in solution and that full-length IRF3 is required for interaction with Npro. Additionally, Nprointeracts with a constitutively active form of IRF3 bound to its transcriptional cofactor, the CREB-binding protein. This is the first study to demonstrate that Nprois able to bind both inactive IRF3 monomer and activated IRF3 dimer and thus likely targets both IRF3 species for ubiquitination and proteasomal degradation.

scholarly journals Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation

2007 ◽  
Vol 81 (7) ◽  
pp. 3087-3096 ◽  
Author(s):  
Oliver Bauhofer ◽  
Artur Summerfield ◽  
Yoshihiro Sakoda ◽  
Jon-Duri Tratschin ◽  
Martin A. Hofmann ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Interferon Regulatory Factor ◽  
Fever Virus ◽  
Target Cells ◽  
Regulatory Factor ◽  
Cmv Promoter ◽  
Interferon Regulatory Factor 3

ABSTRACT Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.

scholarly journals Loss of Interferon Regulatory Factor 3 in Cells Infected with Classical Swine Fever Virus Involves the N-Terminal Protease, Npro

2005 ◽  
Vol 79 (11) ◽  
pp. 7239-7247 ◽  
Author(s):  
S. Anna La Rocca ◽  
Rebecca J. Herbert ◽  
Helen Crooke ◽  
Trevor W. Drew ◽  
Thomas E. Wileman ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Viral Protein ◽  
Sindbis Virus ◽  
Classical Swine Fever ◽  
Interferon Regulatory Factor ◽  
Fever Virus ◽  
Luciferase Reporter ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Infected Cells

ABSTRACT We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (Npro) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from Npro-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.

scholarly journals Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

2008 ◽  
Vol 83 (2) ◽  
pp. 817-829 ◽  
Author(s):  
Nicolas Ruggli ◽  
Artur Summerfield ◽  
Ana R. Fiebach ◽  
Laurence Guzylack-Piriou ◽  
Oliver Bauhofer ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Nonstructural Protein ◽  
Classical Swine Fever ◽  
Interferon Regulatory Factor ◽  
Fever Virus ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Amino Terminal ◽  
Alpha Beta

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

scholarly journals Identification of TRAIL as an Interferon Regulatory Factor 3 Transcriptional Target

2005 ◽  
Vol 79 (14) ◽  
pp. 9320-9324 ◽  
Author(s):  
Jessica R. Kirshner ◽  
Alla Y. Karpova ◽  
Maren Kops ◽  
Peter M. Howley
Keyword(s):  
Viral Infection ◽  
Virus Production ◽  
Interferon Regulatory Factor ◽  
Progeny Virus ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Apoptosis Pathway ◽  
Infected Cells ◽  
Transcriptional Induction ◽  
Transcriptional Target

ABSTRACT Interferon production and apoptosis in virus-infected cells are necessary to prevent progeny virus production and to eliminate infected cells. Paramyxovirus infection induces apoptosis through interferon regulatory factor 3 (IRF-3), but the exact mechanism of how IRF-3 functions is unknown. We show that IRF-3 is involved in the transcriptional induction of TRAIL, a key player in the apoptosis pathway. IRF-3 upregulates TRAIL transcription following viral infection and binds an interferon-stimulated response element in the TRAIL promoter. The mRNA for TRAIL and its receptor, DR5, are induced following viral infection. These studies identify TRAIL as a novel IRF-3 transcriptional target.

Interferon regulatory factor 3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2887-2893 ◽  
Author(s):  
Ezra Aksoy ◽  
Valentina Albarani ◽  
Muriel Nguyen ◽  
Jean-Francois Laes ◽  
Jean-Louis Ruelle ◽  
...  
Keyword(s):  
Cord Blood ◽  
Blood Cells ◽  
Nuclear Translocation ◽  
Interferon Regulatory Factor ◽  
Creb Binding Protein ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Altered Expression ◽  
Neonatal Blood ◽  
Inducible Genes

AbstractThe synthesis of interferon-β (IFNβ) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3–dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNβ and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNβ synthesis because IFNβ signaling was functional in neonatal moDCs. However, addition of exogenous IFNβ failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNβ and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.

scholarly journals Inhibition of Beta Interferon Induction by Severe Acute Respiratory Syndrome Coronavirus Suggests a Two-Step Model for Activation of Interferon Regulatory Factor 3

2005 ◽  
Vol 79 (4) ◽  
pp. 2079-2086 ◽  
Author(s):  
Martin Spiegel ◽  
Andreas Pichlmair ◽  
Luis Martínez-Sobrido ◽  
Jerome Cros ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Virus Infection ◽  
Severe Acute Respiratory Syndrome ◽  
Promoter Activity ◽  
Nuclear Transport ◽  
Interferon Regulatory Factor ◽  
Creb Binding Protein ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3 ◽  
Control Virus ◽  
Novel Coronavirus

ABSTRACT Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. We and others have previously shown that the replication of SARS-CoV can be suppressed by exogenously added interferon (IFN), a cytokine which is normally synthesized by cells as a reaction to virus infection. Here, we demonstrate that SARS-CoV escapes IFN-mediated growth inhibition by preventing the induction of IFN-β. In SARS-CoV-infected cells, no endogenous IFN-β transcripts and no IFN-β promoter activity were detected. Nevertheless, the transcription factor interferon regulatory factor 3 (IRF-3), which is essential for IFN-β promoter activity, was transported from the cytoplasm to the nucleus early after infection with SARS-CoV. However, at a later time point in infection, IRF-3 was again localized in the cytoplasm. By contrast, IRF-3 remained in the nucleus of cells infected with the IFN-inducing control virus Bunyamwera delNSs. Other signs of IRF-3 activation such as hyperphosphorylation, homodimer formation, and recruitment of the coactivator CREB-binding protein (CBP) were found late after infection with the control virus but not with SARS-CoV. Our data suggest that nuclear transport of IRF-3 is an immediate-early reaction to virus infection and may precede its hyperphosphorylation, homodimer formation, and binding to CBP. In order to escape activation of the IFN system, SARS-CoV appears to block a step after the early nuclear transport of IRF-3.

scholarly journals Transcriptional Profiling of Interferon Regulatory Factor 3 Target Genes: Direct Involvement in the Regulation of Interferon-Stimulated Genes

2002 ◽  
Vol 76 (11) ◽  
pp. 5532-5539 ◽  
Author(s):  
Nathalie Grandvaux ◽  
Marc J. Servant ◽  
Benjamin tenOever ◽  
Ganes C. Sen ◽  
Siddarth Balachandran ◽  
...  
Keyword(s):  
Virus Infection ◽  
Target Genes ◽  
Expression Profiles ◽  
Expression System ◽  
Active Form ◽  
Interferon Regulatory Factor ◽  
Inducible Expression ◽  
Immediate Early ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3

ABSTRACT Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-β enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

Sign in / Sign up

Export Citation Format

Share Document