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Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5
Södertörn University, School of Life Sciences, Chemistry. Södertörn University, School of Life Sciences, Molecular biology.
Södertörn University, School of Life Sciences, Chemistry. Södertörn University, School of Life Sciences, Molecular biology.
Södertörn University, School of Life Sciences, Molecular biology.
Södertörn University, School of Life Sciences, Chemistry. Södertörn University, School of Life Sciences, International health.
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, beta PIX 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 beta PIX 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.

Place, publisher, year, edition, pages
2010. Vol. 44, no 3, 260-271 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:sh:diva-6055DOI: 10.1016/j.mcn.2010.03.012ISI: 000278728200006PubMedID: 20363326ScopusID: 2-s2.0-77953231504OAI: oai:DiVA.org:sh-6055DiVA: diva2:395513
Available from: 2011-02-07 Created: 2011-02-07 Last updated: 2016-10-04Bibliographically approved
In thesis
1. 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
2. Molecular characterization of the Tick-borne encephalitis virus: Environments and replication
Open this publication in new window or tab >>Molecular characterization of the Tick-borne encephalitis virus: Environments and replication
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The flavivirus genus is of major concern for world morbidity and mortality and includes viruses causing both encephalitic as well as hemorrhagic diseases. The incidence of Tick-borne encephalitis is increasing in many European countries and several reports have emphasized the expansion of the main vector, Ixodes ricinus. The pattern of vector distribution is also changing in Sweden, which makes it important to set up solid and successful strategies for detection and genetic characterization of novel Swedish TBEV strains.

In this study we have generated strategies for detection of broad types of tick-borne flaviviruses in pools of I. ricinus sampled in Sweden.

The positive collection on the island of Torö was used to generate a sequence of a complete TBEV genome straight from the arthropod reservoir. This cloned virus was used to construct a self-replicating DNA based sub-genomic TBEV replicon capable of expressing reporter genes. The replicon was used to study the effect of TBEV on neurite outgrowth, which revealed that the MTase domain of NS5 block the formation of the Scribble/Rac1/βPIX protein complex, impairing neurite outgrowth in neuronal growth factor induced PC12 cells.

We also demonstrate that TBEV replication is affected by two PDZ binding motifs within NS5 and reveal putative PDZ binding proteins. These interactions might affect cellular pathways and might have a role in flavivirus replication.

We also characterize the variable 3´ non-coding region (V3’-NCR) by in silico studies on TBEV. Analysis brings new evidence that V3’-NCR region carries an enhancer element important for different replication/translation dynamics during the viral lifecycle in mammalian and tick cells. We also propose a temperature-sensitive trans-acting riboswitch mechanism; altering the secondary RNA structures of a closed form at lower temperatures and a form open for translation at higher temperatures. This mechanism may explain the low TBEV level observed in sampled ticks.

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2012. 71 p.
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 63
Keyword
Tick-borne encephalitis virus
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:sh:diva-14829 (URN)978-91-7447-409-1 (ISBN)978-91-86069-42-1 (ISBN)
Public defence
2012-01-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2012-10-30Bibliographically approved

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