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.

The Escherichia coli glutaredoxins 1 and 3 (Grx1 and Grx3) are structurally similar (37% sequence identity), yet have different activities in vivo. Unlike Grx3, Grx1 efficiently reduces protein disulfides in proteins such as RR (ribonucleotide reductase), whereas it is poor at reducing S-glutathionylated proteins. An E. coli strain lacking genes encoding thioredoxins 1 and 2 and Grx1 is not viable on either rich or minimal medium; however, a M43V mutation in Grx3 restores growth under these conditions and results in a Grx1-like protein [Ortenberg, Gon, Porat and Beckwith (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7439-7944]. To uncover the structural basis of this change in activity, we have compared wild-type and mutant Grx3 using CD and NMR spectroscopy. Ligand-induced stability measurements demonstrate that the Grx3(M43V/C65Y) mutant has acquired affinity for RR. Far-UV CD spectra reveal no significant differences, but differences are observed in the near-UV region indicative of tertiary structural changes. NMR 1H- 15N HSQC (heteronuclear single quantum coherence) spectra show that approximately half of the 82 residues experience significant (Δδ > 0.03 p.p.m.) chemical shift deviations in the mutant, including nine residues experiencing extensive (Δδ ≥ 0.15 p.p.m.) deviations. To test whether the M43V mutation alters dynamic properties of Grx3, H/D (hydrogen/deuterium) exchange experiments were performed demonstrating that the rate at which backbone amides exchange protons with the solvent is dramatically enhanced in the mutant, particularly in the core of the protein. These data suggest that the Grx1-like activity of the Grx3(M43V/C65Y) mutant may be explained by enhanced intrinsic motion allowing for increased specificity towards larger substrates such as RR.

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 TBEV-NS5 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.

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.

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-L-glycero-D-manno-heptosyl inner-core moiety (L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glclp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-alpha-Kdop) to which addition of beta-D-Glcp to O-4 of Glcl in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-D-Galp is linked to O-4 of Glcl. In order to test the hypothesis that the 1ex2 locus is involved in the expression Of beta-D-Galp-(1 -> 4-beta-D-Glcp-(1 -> - from Hepl, 1ex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-cleacylated LPS and core oligosaccharide material (OS), as well as ESIMS" on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-D-Glcp extensions from Hepl. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-D-Galp to O-4 of Glcl in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.

We have previously demonstrated that subsets of Ssn6/Tup target genes ave distinct requirements for the Schizosaccharomyces pombe homologs of he Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very igh level of divergence in the histone interacting repression domains f the two proteins suggested that determinants distinguishing Tup11 and up12 might be located in this domain. Here we have combined hylogenetic and structural analysis as well as phenotypic haracterization, under stress conditions that specifically require up12, to identify and characterize the domains involved in up12-specific action. The results indicate that divergence in the epression domain is not generally relevant for Tup12-specific function. nstead, we show that the more highly conserved C-terminal WD40 repeat omain of Tup12 is important for Tup12-specific function. Surface amino cid residues specific for the WD40 repeat domain of Tup12 proteins in ifferent fission yeasts are clustered in blade 3 of the propeller-like tructure that is characteristic of WD40 repeat domains. The Tup11 and up12 proteins in fission yeasts thus provide an excellent model system or studying the functional divergence of WD40 repeat domains.

Interaction between acidic activation domains and the activator-binding domains of Swi1 and Snf5 of the yeast SWI/SNF chromatin remodeling complex has previously been characterized in vitro. Although deletion of both activator-binding domains leads to phenotypes that differ from the wild-type, their relative importance for SWI/SNF recruitment to target genes has not been investigated. In the present study, we used chromatin immunoprecipitation assays to investigate the individual and collective importance of the activator-binding domains for SWI/SNF recruitment to genes within the GAL regulon in vivo. We also investigated the consequences of defective SWI/SNF recruitment for target gene activation. We demonstrate that deletion of both activator-binding domains essentially abolishes galactose-induced SWI/SNF recruitment and causes a reduction in transcriptional activation similar in magnitude to that associated with a complete loss of SWI/SNF activity. The activator-binding domains in Swi1 and Snf5 make approximately equal contributions to the recruitment of SWI/SNF to each of the genes studied. The requirement for SWI/SNF recruitment correlates with GAL genes that are highly and rapidly induced by galactose.

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either L-glycero-D-manno-heptose (LD-Hep) or D-glycero-D-manno-heptose (DD-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode DD-heptosyl- and LD-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.

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.

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.

Non-typeable (acapsular) Haemophilus influenzae (NTHi) is a major cause of otitis media accounting for 25-30% of all cases of the disease. Lipopolysaccharide (LPS) is an essential and exposed component of the H. influenzae cell wall. A characteristic feature of H. influenzae LPS is the extensive inter-strain and intra-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behavior of the bacterium. However, to characterize LPS structure unambiguously is a major challenge due to the extreme heterogeneity of glycoforms that certain strains express. A powerful tool for obtaining sequence and branching information is multiple-stage tandem ESI-MS (ESI-MS(n)) performed on dephosphorylated and permethylated oligosaccharide material using an ESI-quadrupole ion trap mass spectrometer. In general, permethylation increases the MS response by several orders of magnitude and sequence information is readily obtained since methyl tagging allows the distinction between fragment ions generated by cleavage of a single glycosidic bond and inner fragments resulting from the rupture of two glycosidic linkages. Using this approach we are now able to identify all isomeric glycoforms in very heterogeneous LPS preparations.

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 

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.