REGULATION OF HUMAN CYTOMEGALOVIRUS MAJOR IMMEDIATE EARLY GENE EXPRESSION DURING LYTIC REPLICATION

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. Expression of the HCMV major immediate early (MIE) proteins, IE1 and IE2, is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. In Chapter 2 we identified several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to equivalent levels as the known MIE transcript later in infection and were found associated with polyribosomes. These results expand our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Beyond transcriptional regulation, relatively little is known about the post-transcriptional mechanisms that control IE1 and IE2 protein synthesis. In Chapter 3 we found that the canonical MIE 5’ untranslated region (5' UTR) has a positive role in translation control of reporter genes during transfection. We also found that the MIE 5’UTR was necessary for efficient IE1 and IE2 mRNA translation as well as viral replication during infection. These results demonstrate that the shared 5’ UTR of the IE1 and IE2 mRNA is a critical determinant of efficient HCMV replication. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity. In Chapter 4 we applied a kinase capture technique, MIB-MS kinome profiling, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with HCMV. Based on the kinome data, we identified three compounds currently being studied in clinical trials that inhibited HCMV replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs to limit the time and cost of new antiviral drug development.Doctor of Philosoph

Clocks, gradients, and molecular networks: mathematical models for morphogenesis.

The acquisition of a spatial structure during embryo development involves the differentiation of cells, often according to positional information. The complexity of the molecular networks regulating differentiation and of the mechanisms generating positional information makes it necessary to study them by means of mathematical modeling. Vertebrate embryos also acquire a segmented structure during somitogenesis this requires spatial and temporal variations in gene expression, which mathematical modeling can also help understand. A molecular mechanism for the somitogenesis clock is proposed, which accounts for inter-cellular synchronisation, and is based on positive feedback, even though it is compatible with all experimental data interpreted as showing that the clock is based on negative feedback. Experiments proposed to test this model involve real-time clock reporters, as well as inducible systems to induce spatially-controlled perturbations. Theoretical and experimental results have led to conflicting ideas as to how useful positional information can be established. In particular, it has been pointed out that some models of extracellular diffusion of morphogen exhibit inadequate traveling waves of receptor saturation. Two alternative (but not mutually exclusive) models are proposed, which are based on recent experimental results highlighting the roles of extracellular glycoproteins and morphogen oligomerization. The readout of positional information is translated to a discrete set of gene expression patterns. Intriguingly, it has been observed in numerous contexts that genes regulating differentiation are initially co-expressed in progenitors despite their antagonism. We characterise conditions under which three classes of generic "master regulatory networks" can behave as a "multi-switch", directing differentiation in an all-or-none fashion to a specific cell-type chosen among more than two possible outcomes. bHLH dimerisation networks can readily display coexistence of many antagonistic factors when competition is low. Decision-making can be forced by a transient increase in competition, which could correspond to some unexplained experimental observations related to Id proteins

Analyse fonctionnelle du gène BMP-2 lors de la régénération du membre chez l’axolotl

Les amphibiens urodèles (e.g. les axolotls) possèdent la remarquable capacité de régénérer plusieurs parties de leur corps. Ils peuvent, entre autres, régénérer parfaitement un membre amputé par épimorphose, un processus biphasique comprenant une phase de préparation, spécifique à la régénération, et une phase de redéveloppement, commune à l’épimorphose et au développement embryonnaire. Durant la phase de préparation, les cellules du moignon se dédifférencient en cellules pseudo-embryonnaires, prolifèrent et migrent distalement au plan d’amputation pour former un blastème de régénération. Parmi les vertébrés, la dédifférenciation est unique aux urodèles. Afin de mieux comprendre le contrôle moléculaire de la régénération chez les urodèles, nous avons choisi d’étudier BMP-2, un facteur de croissance, en raison de son implication dans la régénération des phalanges distales chez les mammifères. Le facteur de transcription MSX-1 a également été sélectionné en raison de sa capacité à induire la dédifférenciation cellulaire in vitro et de son interaction potentielle avec la signalisation des BMPs. Les résultats présentés dans cette thèse démontrent que BMP-2 et MSX-1 sont exprimés lors des phases de préparation et de redéveloppement de l’épimorphose, et que leur profil d'expression spatio-temporel est très semblable, ce qui suggère une interaction de leurs signaux. En outre, chez les tétrapodes amniotes, l’expression de Shh est restreinte au mésenchyme postérieur des membres en développement et chevauche l’expression de BMP-2. Toutefois, l’expression de BMP-2 n’est pas restreinte à la région postérieure mais forme un gradient postéro-antérieur. Shh est le principal régulateur de la formation du patron de développement antéro-postérieur du ii membre. Étant donné les domaines d’expression chevauchants de BMP-2 et Shh et la restriction postérieure d’expression de Shh, on croit que Shh régule la formation du patron de développement de postérieur à antérieur par l’activation de l’expression de BMP-2. Fait intéressant, l’axolotl exprime également Shh dans la région postérieure, mais le développement des pattes se fait de la région antérieure à la région postérieure au lieu de postérieur à antérieur comme chez les autres tétrapodes, et ceci durant le développement et la régénération. Nous avons utilisé cette caractéristique de l’axolotl pour démontrer que la signalisation Shh ne structure pas l’autopode via BMP-2. En effet, l’expression de BMP-2 n'est pas régulée par l'inhibition de la signalisation Shh, et son expression est du côté opposé à celle de Shh durant le développement et la régénération des pattes de l’axolotl. Il a été observé durant le développement du membre chez la souris que MSX-1 est régulé par la signalisation Shh. Nos résultats ont démontrés que chez l’axolotl, MSX-1 ne semble pas régulé par l'inhibition de la signalisation Shh au cours de la régénération du membre. De plus, nous avons démontré que contrairement à l’expression de Shh, l’expression de BMP-2 est corrélée avec l’ordre de formation des phalanges, est impliquée dans la condensation cellulaire et dans l'apoptose précédant la chondrogenèse. L’ensemble de ces résultats suggère un rôle de BMP-2 dans l’initiation de l’ossification endochondrale. Enfin, nous avons démontré que la signalisation BMP est indispensable pour l’épimorphose du membre durant la phase de redéveloppement.Urodele amphibians (e.g. the axolotls) have a remarkable ability to regenerate parts of their body. They will, among other things, fully regenerate an amputated limb by epimorphosis, a biphasic process comprising a preparation phase, specific to the regeneration, and a redevelopment phase, common to epimorphosis and embryonic development. During the preparation phase, the cells of the stump dedifferentiate into embryonic-like cells, proliferate and migrate distally from the level of amputation to form a regeneration blastema. Among vertebrates, the process of dedifferentiation is unique to urodeles. To better understand the molecular control of regeneration in urodeles, we chose to study BMP-2, a growth factor, because of its involvement in mammalian digit tip regeneration. The transcription factor MSX-1 has also been selected because of its ability to induce cellular dedifferentiation in vitro and its potential interaction with BMPs signaling. The results presented in this thesis show that BMP-2 and MSX-1 are expressed during phases of preparation and redevelopment of epimorphosis, and their spatio-temporal expression profiles are very similar at each stage of epimorphosis, suggesting an interaction of their signals during regeneration. In addition, in tetrapod amniotes, the expression of Shh is restricted to the posterior mesenchyme of developing limbs and overlaps with the expression of BMP-2. However, the expression of BMP-2 is not restricted to the posterior region but forms a posterior-anterior gradient. Shh is the main regulator of the anterior-posterior pattern formation of developing limbs. Given the overlapping expression domains of Shh and BMP-2, and the expression restriction of Shh in posterior, Shh is believed to iv regulate the pattern formation of developing limbs by the activation of BMP-2 expression. Interestingly, the axolotl also expresses Shh in the posterior region, but the limb develops from anterior to posterior rather than posterior to anterior as in other tetrapods, and this, during development and epimorphosis. We used this feature of the axolotl to demonstrate that Shh signaling does not regulate pattern formation through BMP-2. Indeed, the expression of BMP-2 is not regulated by the inhibition of hh signaling, and its expression is opposite to that of Shh during development and regeneration of the axolotl limb. It was observed, during limb development in mice that MSX-1 is regulated by Shh signaling. Our results suggest that in the axolotl, MSX-1 is not regulated by the inhibition of Shh signaling during limb regeneration. Furthermore, we demonstrated that unlike the expression of Shh, the expression of BMP-2 is correlated with the order of formation of the phalanges, is involved in cell condensation and apoptosis preceding chondrogenesis. Taken together, these results suggest a role for BMP-2 in the initiation of endochondral ossification. Finally, we demonstrated that BMP signaling is essential for the redevelopment phase of limb epimorphosis