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Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling.
Södertörn University, School of Life Sciences. Stockholm University.
Södertörn University, School of Life Sciences. Stockholm University.
Södertörn University, School of Life Sciences.
2008 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 10, no 3, 696-712 p.Article in journal (Refereed) Published
Abstract [en]

Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.

Place, publisher, year, edition, pages
2008. Vol. 10, no 3, 696-712 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:sh:diva-6062DOI: 10.1111/j.1462-5822.2007.01076.xISI: 000252897000013PubMedID: 18042258ScopusID: 2-s2.0-38849166538OAI: oai:DiVA.org:sh-6062DiVA: diva2:395518
Available from: 2011-02-07 Created: 2011-02-07 Last updated: 2014-02-27Bibliographically approved
In thesis
1. Functional characterization of interactions between the flavivirus NS5 protein and PDZ proteins of the mammalian host
Open this publication in new window or tab >>Functional characterization of interactions between the flavivirus NS5 protein and PDZ proteins of the mammalian host
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Flaviviruses are found all over the world and affect and infect millions of people every year. Flavivirus infection can lead to severe clinical outcomes resulting in neuronal damages e.g. Tick-borne encephalitis virus (TBEV), or severe hemorrhagic fevers e.g. Dengue virus (DENV). In order to effectively treat infected patients and to prevent these diseases we must understand how these viruses work and how they interfere with the mammalian host. This thesis is focusing on interactions between the virus protein NS5 and human host cell proteins. The interactions presented here might be key factors for out-come of viral disease. NS5 is the largest of the non-structural proteins and is essential for the replication and the capping as it contains both RNA dependent RNA polymerase and Methyltransferase domains. We found that TBEV NS5 interacts with human PDZ domain protein Scribble, a polarization protein important e.g. in regulating membrane trafficking. We determined that the interaction depend on a novel internal motif in TBEVNS5. This interaction could be correlated to NS5s ability to interfere with the immune system as absence of Scribble prevented NS5 from blocking phosphorylation of STAT upon Interferon induction. The role of NS5 in human PDZ domain targeting was addressed further by using a PDZ array system. Both TBEVNS5 and DENVNS5 bind additional PDZ domains using the internal motif. The tight junction protein ZO-1 binds both DENVNS5 and TBEVNS5. DENVNS5 is mainly present in the nucleus and co-localize with ZO-1 in un-polarized cells. In polarized cells TBEVNS5 and ZO-1 co-localize at the plasmamembrane. Putative C-terminal PDZ binding motifs of TBEVNS5 and WNVNS5 were characterized using the PDZ array system. This detected four novel binding partners of TBEVNS5 but numerous of potential WNVNS5 binding partners. We found that TBEVNS5 co-localizes with ZO-2 in the cellular membrane. Further, we found that TBEVNS5 induce the AP-1 by a 2 fold over the control.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2009. 55 p.
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 37
Keyword
Tick-borne encephalitis virus (TBEV), West-Nile virus (WNV), Dengue virus (DENV), flavivirus, PDZ domains, ZO-2, ZO-1, RIMS2, Scribble, Interferon, JAK-STAT signaling, PDZ array, virus-host protein interaction
National Category
Genetics
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:sh:diva-14748 (URN)948-91-86069-05-6 (ISBN)978-91-7155-891-6 (ISBN)
Public defence
2009-06-11, MA 636, Södertörns högskola, Alfred Nobels allé 7, 10:00 (English)
Opponent
Supervisors
Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2012-01-16Bibliographically approved
2. Roles of mammalian Scribble in polarity signaling, virus offense and cell-fate determination
Open this publication in new window or tab >>Roles of mammalian Scribble in polarity signaling, virus offense and cell-fate determination
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5.

TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5.

We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein 

Place, publisher, year, edition, pages
Stockolm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 45 p.
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 47
Keyword
cell polarity, scribble, tbev, RhoGTPase, jak-stat, mapk, neurite outgrowth, influenza a virus
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:sh:diva-14764 (URN)978-91-7447-067-3 (ISBN)978-91-86069-15-5 (ISBN)
Public defence
2010-06-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2012-10-30Bibliographically approved

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