sh.sePublications
Change search
Link to record
Permanent link

Direct link
BETA
Melik, Wessam
Publications (10 of 10) Show all publications
Asghar, N., Lee, Y.-P., Nilsson, E., Lindqvist, R., Melik, W., Kröger, A., . . . Johansson, M. (2016). The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus. Scientific Reports (6), Article ID 39265.
Open this publication in new window or tab >>The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus
Show others...
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, no 6, article id 39265Article in journal (Refereed) Published
Abstract [en]

The tick-borne encephalitis virus (TBEV) is a flavivirus transmitted to humans, usually via tick bites. The virus causes tick-borne encephalitis (TBE) in humans, and symptoms range from mild flu-like symptoms to severe and long-lasting sequelae, including permanent brain damage. It has been suggested that within the population of viruses transmitted to the mammalian host, quasispecies with neurotropic properties might become dominant in the host resulting in neurological symptoms. We previously demonstrated the existence of TBEV variants with variable poly(A) tracts within a single blood-fed tick. To characterize the role of the poly(A) tract in TBEV replication and virulence, we generated infectious clones of Torö-2003 with the wild-type (A)3C(A)6 sequence (Torö-6A) or with a modified (A)3C(A)38 sequence (Torö-38A). Torö-38A replicated poorly compared to Torö-6A in cell culture, but Torö-38A was more virulent than Torö-6A in a mouse model of TBE. Next-generation sequencing of TBEV genomes after passaging in cell culture and/or mouse brain revealed mutations in specific genomic regions and the presence of quasispecies that might contribute to the observed differences in virulence. These data suggest a role for quasispecies development within the poly(A) tract as a virulence determinant for TBEV in mice.

National Category
Biological Sciences Environmental Sciences
Research subject
Environmental Studies
Identifiers
urn:nbn:se:sh:diva-31157 (URN)10.1038/srep39265 (DOI)000389971500001 ()27982069 (PubMedID)2-s2.0-85006377174 (Scopus ID)
Funder
The Foundation for Baltic and East European StudiesKnowledge FoundationSwedish Research CouncilSwedish Foundation for Strategic Research
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-11-16 Created: 2016-11-16 Last updated: 2017-10-30Bibliographically approved
Asghar, N., Lindblom, P., Melik, W., Lindqvist, R., Haglund, M., Forsberg, P., . . . Johansson, M. (2014). Tick-borne encephalitis virus sequenced directly from questing and blood-feeding ticks reveals quasispecies variance.. PLoS ONE, 9(7), Article ID e103264.
Open this publication in new window or tab >>Tick-borne encephalitis virus sequenced directly from questing and blood-feeding ticks reveals quasispecies variance.
Show others...
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, article id e103264Article in journal (Refereed) Published
Abstract [en]

The increased distribution of the tick-borne encephalitis virus (TBEV) in Scandinavia highlights the importance of characterizing novel sequences within the natural foci. In this study, two TBEV strains: the Norwegian Mandal 2009 (questing nymphs pool) and the Swedish Saringe 2009 (blood-fed nymph) were sequenced and phylogenetically characterized. Interestingly, the sequence of Mandal 2009 revealed the shorter form of the TBEV genome, similar to the highly virulent Hypr strain, within the 3' non-coding region (3'NCR). A different genomic structure was found in the 3'NCR of Saringe 2009, as in-depth analysis demonstrated TBEV variants with different lengths within the poly(A) tract. This shows that TBEV quasispecies exists in nature and indicates a putative shift in the quasispecies pool when the virus switches between invertebrate and vertebrate environments. This prompted us to further sequence and analyze the 3'NCRs of additional Scandinavian TBEV strains and control strains, Hypr and Neudoerfl. Toro 2003 and Habo 2011 contained mainly a short (A)3C(A)6 poly(A) tract. A similar pattern was observed for the human TBEV isolates 1993/783 and 1991/4944; however, one clone of 1991/4944 contained an (A)3C(A)11 poly(A) sequence, demonstrating that quasispecies with longer poly(A) could be present in human isolates. Neudoerfl has previously been reported to contain a poly(A) region, but to our surprise the re-sequenced genome contained two major quasispecies variants, both lacking the poly(A) tract. We speculate that the observed differences are important factors for the understanding of virulence, spread, and control of the TBEV.

Keywords
polyadenylic acid, 3' non coding region, adult, article, controlled study, gene sequence, gene structure, gene switching, genetic variability, invertebrate, Ixodes ricinus, nonhuman, nucleotide sequence, nymph, phylogeny, Tick borne encephalitis flavivirus, vertebrate, virus genome, virus strain, virus virulence
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-24381 (URN)10.1371/journal.pone.0103264 (DOI)000341354800074 ()25058476 (PubMedID)2-s2.0-84904787606 (Scopus ID)
Funder
The Foundation for Baltic and East European StudiesKnowledge Foundation
Available from: 2014-08-19 Created: 2014-08-18 Last updated: 2017-12-05Bibliographically approved
Wigerius, M., Asghar, N., Melik, W. & Johansson, M. (2013). Scribble controls NGF-mediated neurite outgrowth in PC12 cells. European Journal of Cell Biology, 92(6-7), 213-221
Open this publication in new window or tab >>Scribble controls NGF-mediated neurite outgrowth in PC12 cells
2013 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 92, no 6-7, p. 213-221Article in journal (Refereed) Published
Abstract [en]

Neurite outgrowth is mediated by dynamic changes of the cytoskeleton and is largely controlled by Rho GTPases and their regulators. Here, we show that the polarity protein Scribble controls PC12 cell neurite outgrowth in response to nerve growth factor. Scribble knockdown decreases neurite numbers and increases neurite length. This effect is linked to TrkA the cognate receptor for NGF as pharmacological inhibition of phosphorylated TrkA (pTrkA) reduces Scribble expression. Moreover, Scribble forms a complex with the MAPK components ERK1/2 in a growth factor dependent manner. In RNAi experiments where Scribble expression is efficiently depleted sustained ERK1/2 phosphorylation is reduced. Conversely, siRNA with intermediate Scribble silencing efficiency fails to match this effect indicating that ERK1/2 activation depends on basic Scribble protein levels. Finally, Scribble translocates to the plasma membrane in response to growth factor where it complexes with HRas and Rac1 suggesting that the phenotype activated by loss of Scribble may be a result of altered GTPase activity. Together, these results demonstrate a novel role for Scribble in neurite outgrowth of PC12 cells.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-19833 (URN)10.1016/j.ejcb.2013.07.002 (DOI)000324971200003 ()23973368 (PubMedID)2-s2.0-84883244028 (Scopus ID)
Funder
The Foundation for Baltic and East European StudiesKnowledge Foundation
Note

Funding agency: Carl Tryggers foundation

Available from: 2013-09-27 Created: 2013-09-27 Last updated: 2017-12-06Bibliographically approved
Bertrand, Y., Töpel, M., Elväng, A., Melik, W. & Johansson, M. (2012). First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses. PLoS ONE, 7(2), e31981
Open this publication in new window or tab >>First Dating of a Recombination Event in Mammalian Tick-Borne Flaviviruses
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2, p. e31981-Article in journal (Refereed) Published
Abstract [en]

The mammalian tick-borne flavivirus group (MTBFG) contains viruses associated with important human and animal diseases such as encephalitis and hemorrhagic fever. In contrast to mosquito-borne flaviviruses where recombination events are frequent, the evolutionary dynamic within the MTBFG was believed to be essentially clonal. This assumption was challenged with the recent report of several homologous recombinations within the Tick-borne encephalitis virus (TBEV). We performed a thorough analysis of publicly available genomes in this group and found no compelling evidence for the previously identified recombinations. However, our results show for the first time that demonstrable recombination (i.e., with large statistical support and strong phylogenetic evidences) has occurred in the MTBFG, more specifically within the Louping ill virus lineage. Putative parents, recombinant strains and breakpoints were further tested for statistical significance using phylogenetic methods. We investigated the time of divergence between the recombinant and parental strains in a Bayesian framework. The recombination was estimated to have occurred during a window of 282 to 76 years before the present. By unravelling the temporal setting of the event, we adduce hypotheses about the ecological conditions that could account for the observed recombination.

National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-17058 (URN)10.1371/journal.pone.0031981 (DOI)000302875500062 ()2-s2.0-84857493482 (Scopus ID)
Funder
The Foundation for Baltic and East European Studies
Available from: 2012-09-13 Created: 2012-09-13 Last updated: 2018-12-17Bibliographically approved
Melik, W. (2012). Molecular characterization of the Tick-borne encephalitis virus: Environments and replication. (Doctoral dissertation). Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University
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. p. 71
Series
Södertörn Doctoral Dissertations, ISSN 1652-7399 ; 63
Keywords
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
Melik, W., Ellencrona, K., Wigerius, M., Elväng, A., Hedström, C. & Johansson, M. (2012). Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication. Virus Research, 169(1), 54-62
Open this publication in new window or tab >>Two PDZ binding motifs within NS5 have roles in Tick-borne encephalitis virus replication
Show others...
2012 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 169, no 1, p. 54-62Article in journal (Refereed) Published
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:sh:diva-14831 (URN)10.1016/j.virusres.2012.07.001 (DOI)000311133800008 ()22796133 (PubMedID)2-s2.0-84867223730 (Scopus ID)
Funder
The Foundation for Baltic and East European StudiesKnowledge Foundation
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2011-12-22 Created: 2012-01-19 Last updated: 2017-12-08Bibliographically approved
Elväng, A., Melik, W., Bertrand, Y., Lönn, M. & Johansson, M. (2011). Sequencing of a Tick-Borne Encephalitis Virus from Ixodes ricinus Reveals a Thermosensitive RNA Switch Significant for Virus Propagation in Ectothermic Arthropods.. Vector Borne and Zoonotic Diseases, 11(6), 649-658
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, p. 649-658Article 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
Microbiology in the medical area
Identifiers
urn:nbn:se:sh:diva-6139 (URN)10.1089/vbz.2010.0105 (DOI)000291717500010 ()21254926 (PubMedID)2-s2.0-79959411565 (Scopus ID)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2018-01-12Bibliographically approved
Wigerius, M., Melik, W., Elväng, A. & Johansson, M. (2010). Rac1 and Scribble are targets for the arrest of neurite outgrowth by TBE virus NS5. Molecular and Cellular Neuroscience, 44(3), 260-271
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, p. 260-271Article 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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:sh:diva-6055 (URN)10.1016/j.mcn.2010.03.012 (DOI)000278728200006 ()20363326 (PubMedID)2-s2.0-77953231504 (Scopus ID)
Available from: 2011-02-07 Created: 2011-02-07 Last updated: 2018-01-12Bibliographically approved
Melik, W., Nilsson, A. S. & Johansson, M. (2007). Detection strategies of tick-borne encephalitis virus in Swedish Ixodes ricinus reveal evolutionary characteristics of emerging tick-borne flaviviruses.. Archives of Virology, 152(5), 1027-1034
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, p. 1027-1034Article 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
Medical and Health Sciences
Identifiers
urn:nbn:se:sh:diva-6063 (URN)10.1007/s00705-006-0922-9 (DOI)000245965300018 ()17277902 (PubMedID)2-s2.0-34247481383 (Scopus ID)
Available from: 2011-02-07 Created: 2011-02-07 Last updated: 2017-12-11Bibliographically approved
Ellencrona, E., Melik, W. & Johansson, M.Novel PDZ dependent cell associations of the NS5 proteins of Tick-borne encephalitis virus and West-Nile virus.
Open this publication in new window or tab >>Novel PDZ dependent cell associations of the NS5 proteins of Tick-borne encephalitis virus and West-Nile virus
(English)Manuscript (preprint) (Other academic)
National Category
Genetics
Identifiers
urn:nbn:se:sh:diva-14752 (URN)
Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2012-01-16Bibliographically approved
Organisations

Search in DiVA

Show all publications