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Roles of mammalian Scribble in polarity signaling, virus offense and cell-fate determination
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Magnus Johansson)
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.
Keyword [en]
cell polarity, scribble, tbev, RhoGTPase, jak-stat, mapk, neurite outgrowth, influenza a virus
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-39368ISBN: 978-91-7447-067-3 (print)OAI: oai:DiVA.org:su-39368DiVA: diva2:319664
Public defence
2010-06-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 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: 2010-05-27 Created: 2010-05-19 Last updated: 2010-05-19Bibliographically approved
List of papers
1. Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling
Open this publication in new window or tab >>Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling
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 relocalization 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
Oxford: Blackwell Publishing Ltd, 2008
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-27642 (URN)10.1111/j.1462-5822.2007.01076.x (DOI)000252897000013 ()
Note
Karin Werme och Michael Wigerius delar första namn på denna artikel. Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2017-12-13Bibliographically approved
2. Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5
Open this publication in new window or tab >>Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5
2010 (English)In: Molecular and Cellular Neuroscience, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 44, no 3, 260-271 p.Article in journal (Refereed) Published
Abstract [en]

Tick-borne encephalitis virus (TBEV) causes extensive CNS disease in humans known as TBE, however, relatively little is known of the molecular mechanisms for its progress. Here, we now show that TBEV produces defects in neuronal development of PC12 cells through a function of the viral NS5 protein. The methyltransferase domain of NS5 is critical and sufficient for restriction of nerve growth factor induced neurite outgrowth. This effect is reversed by expression of NS5 mutants unable to bind Scribble and unexpectedly, in Scribble depleted cells with binding-competent NS5. Furthermore, we also demonstrate that the Rho GTPase Rac1 and the guanine nucleotide-exchange factor, βPIX are outcompeted by NS5 for binding to Scribble, linking to effects on neurite outgrowth by TBEV. Together, these findings provide the first experimental evidence that Rac1 and βPIX are indirect targets of NS5 acting through the multifunctional polarity protein Scribble to oppose neuronal differentiation. In conclusion, our results offer a potential mechanism by which TBEV alters neuronal circuitry and opens new avenues for therapeutic interventions.

Keyword
TBEV, NS5, MTase, Scribble, Neurite morphogenesis, Molecular mechanism of neurotropic, Virus pathogenesis
National Category
Biological Sciences
Research subject
Molecular Genetics; Biochemistry
Identifiers
urn:nbn:se:su:diva-39236 (URN)10.1016/j.mcn.2010.03.012 (DOI)000278728200006 ()
Available from: 2010-05-13 Created: 2010-05-13 Last updated: 2017-12-12Bibliographically approved
3. SCRIBBLE SCAFFOLDS KEY COMPONENTS IN NEURITE OUTGROWTHIMPLICATING DIRECT INVOLVEMENT IN CYTOSKELETON REGULATION
Open this publication in new window or tab >>SCRIBBLE SCAFFOLDS KEY COMPONENTS IN NEURITE OUTGROWTHIMPLICATING DIRECT INVOLVEMENT IN CYTOSKELETON REGULATION
(English)Manuscript (preprint) (Other academic)
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-39237 (URN)
Available from: 2010-05-13 Created: 2010-05-13 Last updated: 2010-05-19Bibliographically approved
4. The NS1 protein of Influenza A virus targets human Scribble in asubtype specific manner
Open this publication in new window or tab >>The NS1 protein of Influenza A virus targets human Scribble in asubtype specific manner
(English)Manuscript (preprint) (Other academic)
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:su:diva-39238 (URN)
Available from: 2010-05-13 Created: 2010-05-13 Last updated: 2010-05-19Bibliographically approved

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