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Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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(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: urn:nbn:se:su:diva-27648OAI: oai:DiVA.org:su-27648DiVA: diva2:216720
Available from: 2011-12-22 Created: 2009-05-11 Last updated: 2011-12-22Bibliographically 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:su:diva-27652 (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, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2009-05-21 Created: 2009-05-11 Last updated: 2009-05-12Bibliographically 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:su:diva-66950 (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, 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

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