Flaviviruses are a group of medically relevant pathogens, known to cause serious disease in animals and humans. The previously defined roles of the flavivirus non-structural protein 2A (NS2A) in RNA replication, and modulation of the host antiviral response, has recently been extended to include virus assembly and secretion. In West Nile virus subtype Kunjin (KUN), an Isoleucine (I)-to-Asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. In this study, prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59, but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59, or the compensatory T149P mutation into NS2A-I59N replicon RNA, restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. These results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for these induced membranes in virus assembly. To further our understanding of how mutations within NS2A are able to affect the induction of virus-induced membranes, leading to a block in virus assembly, the membrane topology of KUN NS2A was investigated. Using a plasmid encoding NS1 and NS2A proteins with C-terminal c-myc and FLAG epitopes, NS2A proteins containing N-linked acceptor sites and C-terminal truncations were generated. Assays were performed to identify the subcellular localization of specific sequences within NS2A by Western blot and immunofluorescence analyses. While the membrane topology could not be determined experimentally, the findings of this study support the assertion that cleavage at the NS1/NS2A junction requires the majority, if not all of the NS2A protein for proper processing to occur, and suggests that the interaction between hydrophilic loops and -helical transmembrane segments plays an important role in the formation and stability of the flavivirus NS2A protein topology. Based on the knowledge of polyprotein processing events, and utilizing a range of software packages, a topology model of NS2A was predicted. The likelihood of additional sequences within NS2A affecting the ability to induce virus-specific membranes, and facilitate virion assembly, has led to the development of an invasive bacterial screening system, as a delivery vehicle to screen libraries of mutated KUN replicon clones. Using these invasive bacteria to deliver mutated KUN replicons into BHK-derived packaging cells, mutations causing a deficiency in either RNA replication or encapsidation can be identified by performing -gal assays on cells maintained in the presence, or absence of Doxycycline (suppressing the expression of structural proteins), respectively. Furthermore, this system was adapted for use in a 96-well plate format, allowing for high-throughput screening. Thus, KUN replicon clones capable of RNA replication, but unable to assemble and secrete virus-like particles can be identified and further analyzed, in the hope of mapping amino acid residues and motifs involved in encapsidation of flavivirus RNA. Finally, a range of hypotheses are discussed, explaining the possible mechanisms by which NS2A is involved in flavivirus assembly. A number of future directions and applications are also presented.

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Der obligat intrazelluläre Parasit Toxoplasma gondii ist der Erreger der Toxoplasmose, und dient zugleich als wichtiger Modellorganismus für weitere Human- und Tierpathogene, wie z.B. Plasmodium oder Eimeria. Die Vermehrung von T. gondii erfordert eine effiziente Biosynthese von Phospholipiden für die Herstellung neuer Membranen, was durch die de novo Synthese durch den Parasiten, und/oder den Import von Lipiden aus der umgebenden Wirtszelle gewährleistet werden kann. Während der Parasit zahlreiche Möglichkeiten für Synthese oder Import von PtdEtn und PtdSer verwendet, scheint die Biosynthese des abundantesten Membranlipids PtdCho auschließlich über den CDP-Cholin Weg zu erfolgen. Dieser erstreckt sich in T. gondii über 3 zelluläre Kompartimente, mit einer cytosolischen Cholin-Kinase (TgCK), einer im Zellkern lokalisierenden Cholin-Cytidylyltransferase (TgCCT) und einer Cholin-Phosphotransferase (TgCPT) im ER. Anders als die substrat-spezifische Ethanolamin-Kinase (TgEK), kann TgCK neben Cholin außerdem Ethanolamin phosphorylieren. TgCK zeigt eine geringe Affinität zu Cholin (Km ~0.77 mM), während eine verkürzte TgCK (TgCKS), welcher eine als Signalpeptid vorhergesagte N-terminale Sequenz (20 Aminosäuren) fehlt, eine etwa 3-fach höhere Aktivität aufweist (Km ~0.26 mM). Während jedoch die Wildtyp-TgCK cytosolische Cluster in Toxoplasma bildet, zeigt die verkürzte TgCK eine gleichmäßigere cytosolische Lokalisierung. Wir schlussfolgern daraus, dass der hydrophobe N-Terminus nicht notwendig ist für eine funktionale TgCK, sondern eine strukturelle Funktion bei der Protein-Lokalisierung hat. Eine konitionelle Mutante, in welcher der TgCK Promoter gegen den Tetracyclin-regulierbaren Promoter pTetO7Sag4 ausgetauscht wurde (Deltatgcki), zeigt erstaunlicherweise normales Wachstum und PtdCho Biosynthese. Die TgCK Aktivität und die daraus resultierende PtdCho Synthese sind nur zu ~30% regulierbar. Unsere Ergebnisse deuten auf die Verwendung eines alternativen Startcodons bzw. Promoters hin, welcher zur Expression einer verkürzten (~53-kDa) aber vermutlich aktiven Cholin Kinase führt, wodurch der Verlust der TgCK (~70-kDa) kompensiert wird. Der konditionelle Knockout von TgCCT, dem regulatorischen Enzym des CDP-Cholin Wegs, hatte einen 50%igen Wachstumsdefekt zur Folge. Diese Studie zeigt eine erstaunliche Flexibilität des Parasiten bezüglich seiner Membranzusammensetzung, und bestätigt zugleich die Annahme, dass PtdCho nicht von der Wirtszelle importiert werden kann. Diese Anpassungsfähigkeit stellt einen möglichen Faktor dar, der es T. gondii erlaubt sich in einem breiten Spektrum von Wirten zu vermehren. / Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes life-threatening disease in neonates and in immunocompromised people. Successful replication of Toxoplasma requires substantial membrane biogenesis, which must be satisfied irrespective of the host-cell milieu. Like in other eukaryotes, the two most abundant phospholipids in the T. gondii membrane are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). Bioinformatics and precursor labeling analyses confirm their synthesis via the CDP-choline and CDP-ethanolamine pathway, respectively. This work shows that the 3-step CDP-choline pathway, involving the activities of TgCK, TgCCT and TgCPT, localizes to the cytosol, nucleus and ER membrane, respectively. The initial reaction is catalyzed by a dual-specificity choline kinase (TgCK, ~70-kDa), capable of phosphorylating choline as well as ethanolamine. The purified full-length TgCK displayed a low affinity for choline (Km ~0.77 mM). TgCK harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. The displacement of the TgCK promoter in a conditional mutant of T. gondii (deltatgcki) attenuated the enzyme expression by ~80%. Unexpectedly, the ?tgcki mutant was not impaired in intracellular growth, and exhibited a normal PtdCho biogenesis. To recompense for the loss of full-length TgCK, the mutant appears to make use of an alternative promoter and/or start codon, resulting in the expression of a shorter but active TgCK isoform identified by the anti-TgCK antiserum, which correlated with its persistent choline kinase activity. Accordingly, the ?tgcki showed an expected incorporation of choline into PtdCho, and susceptibility to dimethylethanolamine (a choline analog). Interestingly, the conditional mutant displayed a regular growth in off state despite a 25% decline in PtdCho content, which suggests a compositional flexibility in T. gondii membranes and insignificant salvage of host-derived PtdCho. The two-step conditional mutagenesis of TgCCT, which caused a reduced growth rate to about 50%, further substantiated this finding. The enzymatic activity of TgCCT and its role in PtdCho synthesis remain to be proven, however. Taken together, the results demonstrate that the CDP-route is likely essential in T. gondii. The competitive inhibition of choline kinase to block the parasite replication appears a potential therapeutic application.The work also reveals a remarkably adaptable membrane biogenesis in T. gondii, which may underly the evolution of Toxoplasma as a promiscuous pathogen.

La protéine BamB est une lipoprotéine de membrane externe appartenant au complexe BAM (β-Barrel Assembly Machinery) et impliquée dans l’assemblage des protéines de membrane externe (PME), la sensibilité aux antibiotiques, le contrôle de l’expression des trois systèmes de sécrétion de type III (T3SS) et la virulence de Salmonella. Chez E. coli, au sein du complexe BAM, elle interagit directement avec la protéine BamA. De plus, chez cette bactérie, BamB présente une activité sérine-thréonine kinase. Afin de mieux caractériser le rôle de BamB, nos objectifs ont été d’étudier (1) l’impact de l’altération de l’interaction de BamB avec le complexe BAM ou de sa séquestration dans le cytoplasme sur l’ensemble des rôles décrits de BamB et (2) l’activité kinase putative de BamB chez Salmonella. Nos résultats montrent que certains rôles de BamB sont dissociables entre eux et que l’interaction BamA/BamB n’est pas requise pour le rôle de BamB dans le contrôle de l’expression des T3SS, la virulence de Salmonella et l’assemblage des PME à la membrane externe. Aucune activité kinase ni aucune activité cytoplasmique de la protéine n’a pu être formellement démontrée. / BamB is an outer-membrane lipoprotein belonging to the BAM complex (β-Barrel Assembly Machinery). In Salmonella, it is involved in the assembly of outer membrane proteins (OMP), in antibiotic susceptibility, in the transcriptional control of the three Type-Three-Secretion-Systems (T3SS) related genes and also in virulence. In E. coli, BamB interacts directly with the BamA protein. Moreover, BamB has been shown to have a serine-threonin kinase activity in this bacterium. In order to better characterize the roles of the BamB protein, our purposes were to study (1) the impact of the alteration of the interaction of BamB with the BAM complex or of its cytoplasmic sequestration and (2) its putative kinase activity in Salmonella. Our results show that some of the BamB roles are dissociable and that the BamA/BamB interaction is not required for T3SS expression, Salmonella virulence or OMP assembly in the outer membrane. Currently, neither a kinase activity nor a cytoplasmic activity has been clearly demonstrated for this protein.