Au cours d'une infection lytique, le virus de l'herpès simplex de type 1 (VHS-1) doit entreprendre plusieurs étapes de fusion afin de se répliquer et se propager correctement. Ainsi, le virus a évolué afin de tirer avantage de la machinerie cellulaire en utilisant des protéines et facteurs de l’hôte à cet effet. Dans la littérature, les processus sous-jacents à l’entrée du VHS-1 ont été largement élucidés. Cependant, on ne sait toujours pas comment les particules virales nouvellement synthétisées sortent de la cellule hôte et quels facteurs cellulaires sont impliqués dans ce processus. Des résultats publiés par notre laboratoire indiquent que la protéine cellulaire, Extended Synaptotagmin 1 (E-Syt1), a un impact négatif sur la propagation globale du virus lorsqu’inhibée par de l’ARN d’interférence. Conséquemment, la présente étude a pour objectif de confirmer et d'approfondir le rôle d’E-Syt1 sur la propagation virale, en particulier sur la sortie du virus. Étant donné que l’activation d’E-Syt1 est liée à l’augmentation de la concentration de calcium cytoplasmique, nous avons également étudié l'implication du calcium au cours des stades ultérieurs de la réplication virale. Ici, nous avons démontré que la surexpression d’E-Syt1 n’a pas d’effet détectable sur la sortie du VHS-1, mais que le calcium a effet sur la propagation virale. Alors que la séquestration précoce du calcium (4 et 6 heures post-infection) à l'aide de chélateurs réprime la sortie virale, aucun effet significatif a été détecté lorsque les chélateurs ont été ajoutés à un stade avancé de l’infection (12 et 16 heures post-infection). Nos résultats fournissent des données intéressantes sur la nécessité de l’homéostasie du calcium intracellulaire afin que VHS-1 puisse assurer une médiation adéquate de la sortie virale. Ces résultats pourraient conduire à la découverte de nouveaux mécanismes ou protéines cellulaires régulées par le calcium et utilisés par le VHS-1 lors de réplications lytiques virales. / During a lytic infection, Herpes Simplex Virus type 1 (HSV-1) must go through multiple steps of fusion to replicate and propagate properly. For this purpose, the virus has evolved consequently by taking advantage of the cellular machinery using host factors and proteins. In the literature, processes underlying HSV-1’s entry have been extensively elucidated. However, it remains unclear how newly synthesized viral particles egress from the host cell, and what cellular factors are implicated in this process. Results published by our laboratory suggest that the cellular protein, Extended Synaptotagmin 1 (E-Syt1), has a negative and global impact on the viral propagation when down regulated by RNA interference. Consequently, this study aims to confirm and deepen our understanding of E-Syt1’s role on HSV-1, particularly during viral egress. Since activation of E-Syt1 is linked to the increase in cytoplasmic calcium concentration, we also investigated calcium involvement during later stages of viral propagation. Interestingly, overexpression of E-Syt1 had no measurable effect on HSV-1 propagation whereas calcium has a dual effect on viral propagation. While early calcium sequestering (4 and 6 hours post-infection) using chelators represses viral egress, no significant effect was detected when chelators were added at later time points (12 and 16 hours post-infection). Our results give interesting insights on how HSV-1 relies on intracellular calcium homeostasis to properly mediate viral egress. These results may lead to the discovery of new mechanisms or cellular proteins that are regulated by calcium and hijacked by HSV-1 during lytic replication.

Die Alzheimer-Krankheit (AD) ist gekennzeichnet durch ein massives Absterben von Neuronen in bestimmten Gehirnregionen. Die zwei charakteristischen histopathologischen Hauptmerkmale sind extrazelluläre Amyloidplaques bestehend aus dem APP-Peptidfragment Abeta und intrazelluläre Fibrillen hyperphosphorylierten Tau-Proteins. Familiäre Formen von AD (FAD) werden verursacht durch Mutationen in den beiden sehr homologen Presenilin-Genen 1 und 2 oder dem APP-Gen. Verschiedene Studien zeigen, dass ein Zusammenhang zwischen Presenilin Mutationen, Abeta-Generierung und Tau-Phosphorylierung beim Auslösen des Neuronentods vorliegt. Immer noch ungeklärt ist, inwiefern Abeta und Presenilin die Tau-abhängige Degeneration beeinflussen. In dieser Arbeit wird gezeigt, dass eine HSV-1-vermittelte Expression von fluoreszenzmarkiertem Tau in kortikalen Primärkulturen einen neurotoxischen Effekt ausübt. Dieser ist drastisch erhöht bei Verwendung eines Konstruktes, welches die pathologische Hyperphosphorylierung von Tau simuliert (pseudohyperphosphoryliertes Tau (PHP-Tau)). Die durch PHP-Tau induzierte Neurodegeneration ist assoziiert mit einer Induktion apoptotischer Mechanismen. Die transgene Expression von wildtyp (wt), aber nicht von FAD-mutiertem PS1 (M146L), unterdrückt PHP-Tau-induzierte Neurodegeneration. Dagegen erhöht die transgene Expression mutierten APPs (SDL) die Degeneration und Phosphorylierung in der Gegenwart von wt, aber nicht von PHP-Tau. Die Daten weisen darauf hin, dass wt und FAD-mutiertes PS1 sowie Abeta die Neurodegeneration durch differentielle Mechanismen modulieren, wobei die Hyperphosphorylierung von Tau entscheidend beteiligt ist. Abeta amplifiziert die Tau-induzierte Neurodegeneration durch die erhöhte Modifikation von Tau. Während PS1 wt der Neurodegeneration durch modifiziertes Tau entgegenwirkt, besitzt FAD-mutiertes PS1 diese Funktion nicht mehr. Demnach könnte die Unterdrückung der Tau-Phosphorylierung eine effektive Therapiemöglichkeit darstellen.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

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Pour compléter leur cycle de vie, les virus interagissent avec de nombreux facteurs de la cellule-hôte. Le virus Herpès simplex de type 1 (HSV-1) ne fait pas exception. Une récente étude protéomique du virus effectuée par notre laboratoire a permis d’identifier 49protéines cellulaires potentiellement incorporées dans les virions matures d’HSV-1 [1]. Étant donné que certaines de ces protéines peuvent jouer des rôles importants au cours du cycle de vie du virus, elles constituent des cibles de choix pour identifier et caractériser de nouvelles interactions hôte-pathogène dans le contexte d’HSV-1. D’ailleurs le laboratoire a été effectué un criblage aux petits ARN d’interférence qui a démontré qu'au moins 15 des protéines incorporées sont impliqués dans le cycle de réplication de HSV-1 en culture cellulaire (Annexe 1). Des nombreuses études rapportent l'incorporation des protéines de l'hôte dans les virions matures mais très peu abordent l'importance de la fraction des protéines cellulaires incorporée dans les virions pour le cycle virale. Pour vérifier ça, nous avons déplété ces protéines des virions matures extracellulaires en utilisant des petits ARN d’interférence. Par la suite, nous avons utilisé ces virus déplétés pour réinfecter des cellules déplétées ou normales. Cette méthode nous a permis d'identifier pour la première fois 8 protéines (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 et 14-3-3ζ) dont l'absence dans les virions réduit la production virale d'au moins 50%. Pour mieux comprendre à quelle étape du cycle viral ces protéines sont nécessaires, nous avons aussi quantifié les virus intracellulaires, produits des cellules déplétées individuellement des quinze protéines cellulaires. Ainsi, nous avons trouvé que dans nos conditions 7 de ces 8 protéines cellulaires (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A et Rab10) semblent impliquées dans la production des virus intracellulaires, ce qui nous a stimulés à débuter une série de tests plus approfondis de l’entrée d’HSV-1. Les résultats préliminaires, démontrent l’implication dans l’entrée d’HSV-1 d’au moins 3 à 4 de ces protéines (HSPA8, KRT10, Rab5A et Rab10). / To complete their life cycle viruses interact with many factors of the host cell. Herpes simplex virus type 1 (HSV-1) is no exception. A recent proteomic study of the virus carried by our laboratory has identified up to 49 cellular proteins potentially incorporated into the mature virions of HSV-1[1]. Since some of these proteins may play important roles during the viral life cycle, they are interesting targets for identification and characterization of new host-pathogen interactions in the context of HSV-1. To target the proteins that are relevant to the viral life cycle of Herpes, the laboratory performed a screening with small interfering RNAs (siRNAs), which showed that at least 15 incorporated proteins are involved in the replication cycle of HSV- 1 in cell culture (Appendix 1). Numerous studies report the incorporation of host proteins in mature virions but few addresses the importance for the viral infectivity of the fractions of cellular proteins incorporated into the virions. To verify this, we depleted these proteins from the mature extracellular virions using siRNAs. Subsequently, we used these viruses to re-infect depleted or normal cells. This method allowed us to identify for the first time eight proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 and 14-3-3ζ) whose absence in virions reduced viral production by at least 50%. As part of understanding at what stage of the life cycle these proteins are necessary for HSV-1, we tested the infectivity of intracellular depleted viruses. Thus, we found at least seven cellular proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A and Rab10) to have a pronounced effect on the replication of herpes virus, which has stimulated us to begin a series of more in-depth tests of the entry of HSV-1. Preliminary results demonstrate the involvement in the entry of HSV-1 of at least three to four proteins (HSPA8, KRT10, Rab5A and Rab10).

Herpesviruses can enter host cells by pH-dependent endocytic pathways in a cell-specific manner. The role of pH in herpesvirus endocytosis is unclear. Herpes simplex virus (HSV) is a paradigm for virus membrane fusion via a complex of glycoproteins. HSV glycoproteins B, D and the heterodimer H-L are necessary and sufficient for membrane fusion. This work analyzes the structure and function of HSV glycoproteins B, D, and H-L at neutral pH, and at the physiological low-pH encountered during endocytic entry. It is demonstrated that mildly acidic low pH triggers specific conformational changes in HSV gB at a pH of 5.7 to 6.0. The antigenic structure of gB functional region I that is critical for fusion is specifically altered by mildly acidic pH both in vitro and during entry into host cells. Point mutations within gB functional region 1 that block membrane fusion still allow conformational changes in region 1. This suggests that specific hydrophobic residues are essential for fusion domain insertion into the host cell membrane but not conformational change. The detected conformational changes were reversible, similar to other class III fusion glycoproteins. Exposure to mildly acidic pH directly triggered the fusion function of HSV glycoproteins and caused gB, but not other glycoproteins, to become more hydrophobic. The oligomeric conformation of gB is altered at a similar pH range. In addition, several approaches were used to monitor gB throughout glycoprotein synthesis and maturation. It is shown that gB may cotranslationally fold and oligomerize as it is synthesized on the ribosome. As gB matures it then alters conformation and/or binding partner to form antigenically distinct populations of gB within the cell and virion. I conclude that intracellular low pH induces changes in gB conformation that, together with additional triggers such as receptor-binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity.

Pour compléter leur cycle de vie, les virus interagissent avec de nombreux facteurs de la cellule-hôte. Le virus Herpès simplex de type 1 (HSV-1) ne fait pas exception. Une récente étude protéomique du virus effectuée par notre laboratoire a permis d’identifier 49protéines cellulaires potentiellement incorporées dans les virions matures d’HSV-1 [1]. Étant donné que certaines de ces protéines peuvent jouer des rôles importants au cours du cycle de vie du virus, elles constituent des cibles de choix pour identifier et caractériser de nouvelles interactions hôte-pathogène dans le contexte d’HSV-1. D’ailleurs le laboratoire a été effectué un criblage aux petits ARN d’interférence qui a démontré qu'au moins 15 des protéines incorporées sont impliqués dans le cycle de réplication de HSV-1 en culture cellulaire (Annexe 1). Des nombreuses études rapportent l'incorporation des protéines de l'hôte dans les virions matures mais très peu abordent l'importance de la fraction des protéines cellulaires incorporée dans les virions pour le cycle virale. Pour vérifier ça, nous avons déplété ces protéines des virions matures extracellulaires en utilisant des petits ARN d’interférence. Par la suite, nous avons utilisé ces virus déplétés pour réinfecter des cellules déplétées ou normales. Cette méthode nous a permis d'identifier pour la première fois 8 protéines (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 et 14-3-3ζ) dont l'absence dans les virions réduit la production virale d'au moins 50%. Pour mieux comprendre à quelle étape du cycle viral ces protéines sont nécessaires, nous avons aussi quantifié les virus intracellulaires, produits des cellules déplétées individuellement des quinze protéines cellulaires. Ainsi, nous avons trouvé que dans nos conditions 7 de ces 8 protéines cellulaires (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A et Rab10) semblent impliquées dans la production des virus intracellulaires, ce qui nous a stimulés à débuter une série de tests plus approfondis de l’entrée d’HSV-1. Les résultats préliminaires, démontrent l’implication dans l’entrée d’HSV-1 d’au moins 3 à 4 de ces protéines (HSPA8, KRT10, Rab5A et Rab10). / To complete their life cycle viruses interact with many factors of the host cell. Herpes simplex virus type 1 (HSV-1) is no exception. A recent proteomic study of the virus carried by our laboratory has identified up to 49 cellular proteins potentially incorporated into the mature virions of HSV-1[1]. Since some of these proteins may play important roles during the viral life cycle, they are interesting targets for identification and characterization of new host-pathogen interactions in the context of HSV-1. To target the proteins that are relevant to the viral life cycle of Herpes, the laboratory performed a screening with small interfering RNAs (siRNAs), which showed that at least 15 incorporated proteins are involved in the replication cycle of HSV- 1 in cell culture (Appendix 1). Numerous studies report the incorporation of host proteins in mature virions but few addresses the importance for the viral infectivity of the fractions of cellular proteins incorporated into the virions. To verify this, we depleted these proteins from the mature extracellular virions using siRNAs. Subsequently, we used these viruses to re-infect depleted or normal cells. This method allowed us to identify for the first time eight proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A, Rab10 and 14-3-3ζ) whose absence in virions reduced viral production by at least 50%. As part of understanding at what stage of the life cycle these proteins are necessary for HSV-1, we tested the infectivity of intracellular depleted viruses. Thus, we found at least seven cellular proteins (DDX3X, HSPA8, KRT10, MIF, Rab5A, Rab6A and Rab10) to have a pronounced effect on the replication of herpes virus, which has stimulated us to begin a series of more in-depth tests of the entry of HSV-1. Preliminary results demonstrate the involvement in the entry of HSV-1 of at least three to four proteins (HSPA8, KRT10, Rab5A and Rab10).

Herpesviruses have co-speciated with several vertebrate and invertebrate animals throughout the history of evolution. In the immunocompetent human host, primary infection is usually benign, whereafter the virus is brought into life-long latency. Viral reactivation can however cause severe disease in immunocompromised, and rarely also in immunocompetent, patients. The overall aim of this thesis was to study the immunologic effects of cytomegalovirus (CMV) and herpes simplex type 1 (HSV-1) infection in neurocognitive disorders. CMV is known to promote T-cell differentiation towards a more effector-oriented phenotype, similar to what is seen in the elderly. We have addressed the frequency of CMV-specific CD8+ T-cells in Alzheimer's disease (AD). Furthermore, we have investigated whether AD patients present with a different CMV-specific immune profile, overall CD8 phenotype or inflammatory cytokine response to anti-CD3/CD28 beads, CMV pp65 and amyloid beta. Subjects with AD presented with a lower proportion of CMV-specific CD8+ T-cells compared to non-demented (ND) controls, but no differences in overall CD8 differentiation were seen. Overall, AD subjects presented with a more pro-inflammatory peripheral blood mononuclear cell (PBMC) phenotype. When PBMCs were challenged with CD3/CD28-stimulation, CMV seropositive AD subjects presented with more IFN-γ release than both CMV seronegative AD subjects and CMV seropositive ND controls. For effective screening of humoral herpesvirus immunity, both in research and in clinical practice, efficient immunoassays are needed. We have addressed the methodology of multiplex herpesvirus immunoassays and related bioinformatics and investigated antibody levels in AD patients and ND controls. Subjects with AD presented with lower levels of human herpesvirus 6 (HHV-6) IgG. However, there was no difference in HHV-6 DNA levels in PBMCs between the groups. Herpes simplex encephalitis (HSE) is a devastating disease, where antiviral treatment has greatly decreased mortality but not eliminated the associated long-term neurocognitive morbidity. We have investigated the correlation between N-Methyl-D-Aspartate Receptor (NMDAR) autoimmunity and recovery of neurocognitive functions after HSE. Approximately one quarter of all HSE cases developed NMDAR autoantibodies within 3 months after onset of disease. Antibody development was associated with an impaired neurocognitive recovery during the two year follow-up and could become an important therapy guiding factor in the future.