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Molecular characterization of the Tick-borne encephalitis virus: Environments and replication
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. (Dr Magnus Johansson (SH))
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 [en]
Tick-borne encephalitis virus
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Genetics
Identifiers
URN: urn:nbn:se:su:diva-66950ISBN: 978-91-7447-409-1 (print)ISBN: 978-91-86069-42-1 (print)OAI: oai:DiVA.org:su-66950DiVA: diva2:469026
Public defence
2012-01-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 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: 2013-04-09 Created: 2011-12-22 Last updated: 2013-04-09Bibliographically approved
List of papers
1. Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses
Open this publication in new window or tab >>Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses
2007 (English)In: Archives of Virology, ISSN 0304-8608, E-ISSN 1432-8798, Vol. 152, no 5, 1027-1034 p.Article in journal (Refereed) Published
Abstract [en]

The flaviviral tick-borne encephalitis virus (TBEV) is a human pathogen having significant impact on public health. The geographical distribution of TBEV and TBEV-like viruses is increasing, which makes it important to characterise the natural virus populations. Here we present four RT-PCR strategies designed for detection of broad types of tick-borne flaviviruses. Sequence information on more than 32% of a TBEV genome was generated from a small pool of ticks collected in the Stockholm archipelago on the island of Torö. The sequences were characterised and compared with those of other tick-borne flaviviruses, which classified the virus as Western European TBEV.

National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-16115 (URN)10.1007/s00705-006-0922-9 (DOI)000245965300018 ()17277902 (PubMedID)
Available from: 2007-10-03 Created: 2007-10-03 Last updated: 2011-12-22Bibliographically approved
2. Sequencing of a Tick-Borne Encephalitis Virus from Ixodes ricinus Reveals a Thermosensitive RNA Switch Significant for Virus Propagation in Ectothermic Arthropods
Open this publication in new window or tab >>Sequencing of a Tick-Borne Encephalitis Virus from Ixodes ricinus Reveals a Thermosensitive RNA Switch Significant for Virus Propagation in Ectothermic Arthropods
Show others...
2011 (English)In: Vector Borne and Zoonotic Diseases, ISSN 1530-3667, E-ISSN 1557-7759, Vol. 11, no 6, 649-658 p.Article in journal (Refereed) Published
Abstract [en]

Tick-borne encephalitis virus (TBEV) is a flavivirus with major impact on global health. The geographical TBEV distribution is expanding, thus making it pivotal to further characterize the natural virus populations. In this study, we completed the earlier partial sequencing of a TBEV pulled out of a pool of RNA extracted from 115 ticks collected on Torö in the Stockholm archipelago. The total RNA was sufficient for all sequencing of a TBEV genome (Torö-2003), without conventional enrichment procedures such as cell culturing or suckling mice amplification. To our knowledge, this is the first time that the genome of TBEV has been sequenced directly from an arthropod reservoir. The Torö-2003 sequence has been characterized and compared with other TBE viruses. In silico analyses of secondary RNA structures formed by the two untranslated regions revealed a temperature-sensitive structural shift between a closed replicative form and an open AUG accessible form, analogous to a recently described bacterial thermoswitch. Additionally, novel phylogenetic conserved structures were identified in the variable part of the 3′-untranslated region, and their sequence and structure similarity when compared with earlier identified structures suggests an enhancing function on virus replication and translation. We propose that the thermo-switch mechanism may explain the low TBEV prevalence often observed in environmentally sampled ticks. Finally, we were able to detect variations that help in the understanding of virus adaptations to varied environmental temperatures and mammalian hosts through a comparative approach that compares RNA folding dynamics between strains with different mammalian cell passage histories.

National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-67054 (URN)10.1089/vbz.2010.0105 (DOI)000291717500010 ()
Note
authorCount :5Available from: 2011-12-22 Created: 2011-12-22 Last updated: 2017-12-08Bibliographically approved
3. 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
4. Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication
Open this publication in new window or tab >>Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication
Show others...
(English)Manuscript (Other academic)
Abstract [en]

The flavivirus genus includes important human pathogens like Tick-borne encephalitis virus (TBEV), Dengue virus (DV) and West-Nile virus (WNV), that can cause severe disease e.g. encephalitis or hemorrhagic fever. The NS5 protein is a multifunctional RNA dependent RNA polymerase indispensable for the flavivirus replication. We have previously shown that TBEVNS5 contains a unique internal PDZ binding motif (YS223) for specific targeting of the PDZ protein Scribble. This interaction has impact on both viral down regulation of host cellular defense systems and neurite outgrowth. Putative C-terminal PDZ binding motifs present in TBEVNS5 (-SII903) and WNVNS5 (-TVL905) have also previously been highlighted.

To determine whether the PDZ binding motifs of TBEVNS5 has an effect on virus replication we constructed a DNA based sub-genomic TBEV replicon expressing firefly luciferase. The motifs within NS5 were mutated individually and in concert and the replicons were assayed in cell culture. Our results show that the replication rate was impaired in all mutants, which indicates that PDZ dependent host interactions influence flavivirus replication.We also find that the C-terminal PDZ binding motif present in TBEVNS5 and WNVNS5 are targeting various human PDZ domain proteins. TBEVNS5 has high affinity to Zonulaoccludens-2 (ZO-2),GIAP C-terminus interacting protein (GIPC), Calcium/calmodulin-dependent serine protein kinase (CASK) and Interleukin 16 (IL-16).A different pattern was observed for WNVNS5 as it associated with IL-16, and several other putative interaction partners.

National Category
Biological Sciences
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-27648 (URN)
Available from: 2011-12-22 Created: 2009-05-11 Last updated: 2011-12-22Bibliographically approved

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