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1.
PLoS Pathog ; 14(4): e1006980, 2018 04.
Article in English | MEDLINE | ID: mdl-29709033

ABSTRACT

Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.


Subject(s)
Antiviral Agents/pharmacology , Gene Expression Regulation/drug effects , Influenza, Human/immunology , Thogotovirus/immunology , Transcription Factor TFIIB/metabolism , Transcription, Genetic/drug effects , Viral Proteins/metabolism , HeLa Cells , Humans , Influenza, Human/drug therapy , Influenza, Human/metabolism , Promoter Regions, Genetic , Thogotovirus/drug effects , Transcription Factor TFIIB/genetics
2.
J Virol ; 84(23): 12344-50, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20861272

ABSTRACT

Type I interferon (IFN-α/ß) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.


Subject(s)
DEAD-box RNA Helicases/immunology , Dendritic Cells/immunology , Interferon Type I/immunology , Orthomyxoviridae Infections/immunology , Signal Transduction/immunology , Thogotovirus/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Vesicular Transport/genetics , Animals , DEAD Box Protein 58 , Interferon Type I/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Immunological , Myeloid Differentiation Factor 88/genetics , Reverse Transcriptase Polymerase Chain Reaction
3.
J Virol ; 84(15): 7634-41, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20484517

ABSTRACT

The highly pathogenic Nipah virus (NiV) is aerially transmitted and causes a systemic infection after entering the respiratory tract. Airway epithelia are thus important targets in primary infection. Furthermore, virus replication in the mucosal surfaces of the respiratory or urinary tract in later phases of infection is essential for virus shedding and transmission. So far, the mechanisms of NiV replication in epithelial cells are poorly elucidated. In the present study, we provide evidence that bipolar targeting of the two NiV surface glycoproteins G and F is of biological importance for fusion in polarized epithelia. We demonstrate that infection of polarized cells induces focus formation, with both glycoproteins located at lateral membranes of infected cells adjacent to uninfected cells. Supporting the idea of a direct spread of infection via lateral cell-to-cell fusion, we could identify basolateral targeting signals in the cytoplasmic domains of both NiV glycoproteins. Tyrosine 525 in the F protein is part of an endocytosis signal and is also responsible for basolateral sorting. Surprisingly, we identified a dityrosine motif at position 28/29 in the G protein, which mediates polarized targeting. A dileucine motif predicted to function as sorting signal is not involved. Mutation of the targeting signal in one of the NiV glycoproteins prevented the fusion of polarized cells, suggesting that basolateral or bipolar F and G expression facilitates the spread of NiV within epithelial cell monolayers, thereby contributing to efficient virus spread in mucosal surfaces in early and late phases of infection.


Subject(s)
Epithelial Cells/virology , Nipah Virus/physiology , Tyrosine/metabolism , Viral Envelope Proteins/physiology , Virus Internalization , Virus Replication , Amino Acid Motifs , Animals , Cell Fusion , Endocytosis , Humans
4.
J Gen Virol ; 91(Pt 1): 220-7, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19812269

ABSTRACT

The tick-transmitted orthomyxovirus Thogoto virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.


Subject(s)
Interferon Regulatory Factor-7/antagonists & inhibitors , Thogotovirus/immunology , Thogotovirus/pathogenicity , Viral Proteins/physiology , Virulence Factors/physiology , Animals , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/virology , Dimerization , Fibroblasts/immunology , Fibroblasts/virology , Interferon Regulatory Factor-3/antagonists & inhibitors , Interferon-gamma/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Protein Binding , Protein Interaction Mapping , TNF Receptor-Associated Factor 6/metabolism
5.
J Virol ; 82(22): 11446-53, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18768974

ABSTRACT

The ML protein of Thogoto virus, a tick-transmitted orthomyxovirus, is a splice variant of the viral matrix protein and antagonizes the induction of antiviral type I interferon (IFN). Here we identified the general RNA polymerase II transcription factor IIB (TFIIB) as an ML-interacting protein. Overexpression of TFIIB neutralized the inhibitory effect of ML on IRF3-mediated promoter activation. Moreover, a recombinant virus expressing a mutant ML protein unable to bind TFIIB was severely impaired in its ability to suppress IFN induction. We concluded that TFIIB binding is required for the IFN antagonist effect exerted by ML. We further demonstrate that the ML-TFIIB interaction has surprisingly little impact on gene expression in general, while a strong negative effect is observed for IRF3- and NF-kappaB-regulated promoters.


Subject(s)
Interferon Regulatory Factor-3/antagonists & inhibitors , Thogotovirus/physiology , Transcription Factor TFIIB/metabolism , Viral Proteins/metabolism , Cell Line , Humans , Protein Interaction Mapping , Thogotovirus/immunology
6.
J Virol ; 79(6): 3865-72, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15731282

ABSTRACT

Nipah virus (NiV), a highly pathogenic member of the family Paramyxoviridae, encodes the surface glycoproteins F and G. Since internalization of the NiV envelope proteins from the cell surface might be of functional importance for viral pathogenesis either by regulating cytopathogenicity or by modulating recognition of infected cells by the immune system, we analyzed the endocytosis of the NiV F and G proteins. Interestingly, we found both glycoproteins to be internalized in infected and transfected cells. As endocytosis is normally mediated by tyrosine- or dileucine-dependent signals in the cytoplasmic tails of transmembrane proteins, all potential internalization signals in the NiV glycoproteins were mutated. Whereas the G protein appeared to be constitutively internalized with the bulk flow during membrane turnover, uptake of the F protein was found to be signal mediated. F endocytosis clearly depended on a membrane-proximal YXXPhi motif and was found to be of functional importance for the biological activity of the protein.


Subject(s)
Endocytosis , Glycoproteins/metabolism , Nipah Virus/metabolism , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Dogs , Glycoproteins/chemistry , Glycoproteins/genetics , Leucine/genetics , Leucine/physiology , Molecular Sequence Data , Mutation , Nipah Virus/genetics , Tyrosine/genetics , Tyrosine/physiology , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics
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