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Functional characterization of interactions between the flavivirus NS5 protein and PDZ proteins of the mammalian host
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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 [en]
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: urn:nbn:se:su:diva-27652ISBN: 948-91-86069-05-6 ISBN: 978-91-7155-891-6 (print)OAI: oai:DiVA.org:su-27652DiVA: diva2:216738
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
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. Flavivirus NS5 associates with host-cell proteins zonula occludens-1 (ZO-1) and regulating synaptic membrane exocytosis-2 (RIMS2) via an internal PDZ binding mechanism
Open this publication in new window or tab >>Flavivirus NS5 associates with host-cell proteins zonula occludens-1 (ZO-1) and regulating synaptic membrane exocytosis-2 (RIMS2) via an internal PDZ binding mechanism
2009 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 390, 319-323 p.Article in journal (Refereed) Published
Abstract [en]

Dengue virus (DENV) and tick-borne encephalitis virus (TBEV) are flaviviruses, which can cause lethal hemorrhagic fever and encephalitis, respectively. Here, we demonstrate that the TBEV-NS5 and DENV-NS5 proteins use an internal binding mechanism to target human PDZ proteins. TBEV-NS5 has high affinity to regulating synaptic membrane exocytosis-2 (RIMS2) and Scribble, whereas DENV-NS5 binds primarily to the tight junction protein zonula occludens-1 (ZO-1). Targeting of TBEVNS5 to the plasma membrane is stabilised by ZO-1; however, DENV-NS5 co-localises with ZO-1 in the nucleus. These interactions have potential important roles in the ability of flaviviruses to manipulate cell proliferation, junction permeability and the interferon pathways.

Place, publisher, year, edition, pages
Berlin: Walter de Gruyter, 2009
Research subject
Molecular Genetics
Identifiers
urn:nbn:se:su:diva-27644 (URN)10.1515/BC.2009.041 (DOI)000265160000005 ()
Available from: 2009-05-11 Created: 2009-05-11 Last updated: 2017-12-13Bibliographically approved
3. 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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