Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope and a proteinaceous tegument layer. There are over fifty viral proteins and many host proteins identified in HSV-1 virions. The final formation of mature virus particles requires the membrane wrapping of tegumented capsids in the cytoplasm, a process termed secondary envelopment. This process involves the coordination of numerous viral and cellular proteins and results in double-membrane structures with enveloped virions contained within cellular vesicles. Mature viruses are then released through the fusion of these virion-containing vesicles and plasma membranes. This thesis describes investigation into the functions of viral glycoprotein M (gM) and the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) in secondary envelopment. Firstly, it has been reported that gH/L can be efficiently internalised and targeted to the TGN by the co-expression of gM in transfection assays. In order to examine the role of gM in guiding the localisation of viral proteins in infected cells, a HSV-1 gM deletion virus (∆gM), and its revertant virus were constructed. The major phenotype demonstrated was that the absence of gM caused the internalisation of cell surface gH/L to be inhibited and higher levels of gH/L to be observed on the cell surface. Further, lower levels of gH/L were detected in purified ∆gM virions, which was in agreement with the delayed entry kinetics, smaller plaque sizes and greater replication deficits at low multiplicity of infection observed in ∆gM infected cells. Over all the results presented in this thesis demonstrate that in infected cells the efficient incorporation of gH/L into virions relies on the function of gM in HSV-1. Secondly, during HSV-1 secondary envelopment the budding and scission of the viral envelope from the host membrane share topological similarities with the formation of intraluminal vesicle in multivesicular bodies, retrovirus budding, and abscission at the end of cytokinesis, processes that require the cellular ESCRT machinery. There are four multiprotein ESCRT complexes and many associated proteins involved in their regulation. It has been previously shown that the ESCRT-III complex and a functional ATPase VPS4 are required for HSV-1 secondary envelopment, but different from the strategy utilised by HIV-1, the recruitment of ESCRT during HSV-1 infection is independent of TSG101 and/or ALIX. Data presented in this thesis demonstrate that CHMP4A/B/C proteins of the ESCRT-III complex are specifically crucial for HSV-1 secondary envelopment. Simultaneous depletion of CHMP4A/B/C proteins significantly inhibited HSV-1 replication. Ultrastructure analysis revealed that there were virtually no extracellular virions in CHMP4A/B/C depleted samples while more free capsids were observed in the cytoplasm, although the nuclear capsids and primary envelopment events appeared to be normal. In order to identify interactions between HSV-1 and ESCRT proteins, 22 HSV-1 tegument proteins were cloned and tested against a panel of ESCRT and ESCRT-associated proteins in yeast two-hydrid assays. Analysis of positive hits from yeast two-hybrid interaction screens using GST pull-down, co-immunoprecipitation and protein co-localisation assays have validated interactions of pUL47 with CC2D1A/1B, CIN85, CHMP6 and ALIX, pUL46 and pUL49 with CC2D1A/1B and CIN85, and pUL16 with CC2D1A/1B. Furthermore, the newly identified ESCRT associated proteins CC2D1A and CC2D1B have been detected in purified virions. The role of the identified ESCRT proteins in HSV-1 replication has been investigated using siRNA depletion. Unfortunately siRNA depletions of the various ESCRT candidates individually or in combinations did not show any significant effect on HSV-1 replication. Overall these data suggest that unlike HIV and other retroviruses, HSV-1 has evolved multiple parallel pathways to hijack the ESCRT machinery to facilitate its replication, particularly, through the interactions that lead directly to the recruitment of CHMP4A/B/C proteins. Disruption of some of these pathways did not prevent HSV-1 replication in tissue culture, suggesting any one potential pathway is sufficient for ESCRT recruitment to sites of HSV-1 assembly.

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.

Le Virus Herpès Simplex de type 1 (HSV-1) est un agent infectieux qui cause l’herpès chez une grande proportion de la population mondiale. L’herpès est généralement considéré comme une maladie bénigne dont la forme la plus commune est l'herpès labial (communément appelé « bouton de fièvre »), mais elle peut se révéler très sérieuse et causer la cécité et l’encéphalite, voir létale dans certain cas. Le virus persiste toute la vie dans le corps de son hôte. Jusqu'à présent, aucun traitement ne peut éliminer le virus et aucun vaccin n’a été prouvé efficace pour contrôler l’infection herpétique. HSV-1 est un virus avec un génome d’ADN bicaténaire contenu dans une capside icosaèdrale entourée d’une enveloppe lipidique. Treize glycoprotéines virales se trouvent dans cette enveloppe et sont connues ou supposées jouer des rôles distincts dans différentes étapes du cycle de réplication viral, incluant l'attachement, l'entrée, l’assemblage, et la propagation des virus. La glycoprotéine M (gM) qui figure parmi ces glycoprotéines d’enveloppe, est la seule glycoprotéine non essentielle mais est conservée dans toute la famille herpesviridae. Récemment, l’homologue de gM dans le Pseudorabies virus (PRV), un autre herpesvirus, a été impliqué dans la phase finale de l’assemblage (i.e. l’enveloppement cytoplasmique) au niveau du réseau trans-Golgi (TGN) en reconnaissant spécifiquement des protéines tégumentaires et d’autres glycoprotéines d’enveloppe ([1]). Toutefois, il a été proposé que cette hypothèse ne s’applique pas pour le HSV-1 ([2]). De plus, contrairement à la localisation au TGN dans les cellules transfectées, HSV-1 gM se localise dans la membrane nucléaire et sur les virions périnucléaires durant une infection. L’objectif du projet présenté ici était d’éclaircir la relation de la localisation et la fonction de HSV-1 gM dans le contexte d’une infection. Dans les résultats rapportés ici, nous décrivons tout abord un mécanisme spécifique de ciblage nucléaire de HSV-1 gM. En phase précoce d’une infection, gM est ciblée à la membrane nucléaire d'une manière virus ii dépendante. Cela se produit avant la réorganisation du TGN normalement induite par l’infection et avant que gM n’entre dans la voie de sécrétion. Ce ciblage nucléaire actif et spécifique de gM ne semble pas dépendre des plusieurs des partenaires d’interaction proposés dans la littérature. Ces données suggèrent que la forme nucléaire de gM pourrait avoir un nouveau rôle indépendant de l’enveloppement final dans le cytoplasme. Dans la deuxième partie du travail présenté ici, nous avons concentré nos efforts sur le rôle de gM dans l’assemblage du virus en phase tardive de l’infection et en identifiant un domaine critique de gM. Nos résultats mettent en valeur l’importance du domaine carboxyl-terminal cytoplasmique de gM dans le transport de gM du réticulum endoplasmique (RE) à l’appareil de Golgi, dans l’enveloppement cytoplasmique et la propagation intercellulaire du virus. Ainsi, l’export du RE de gM a été complètement compromis dans les cellules transfectées exprimant un mutant de gM dépourvu de sa région C-terminale. La délétion la queue cytoplasmique de gM cause une réduction légère du titre viral et de la taille des plaques. L'analyse de ces mutants par microscopie électronique a démontré une accumulation des nucléocapsides sans enveloppe dans le cytoplasme par rapport aux virus de type sauvage. Étrangement, ce phénotype était apparent dans les cellules BHK mais absent dans les cellules 143B, suggérant que la fonction de gM dépende du type cellulaire. Finalement, le criblage de partenaires d’interaction du domaine C-terminal de gM identifiés par le système de double-hybride nous a permis de proposer plusieurs candidats susceptibles de réguler la fonction de gM dans la morphogénèse et la propagation de virus. / Herpes Simplex Virus type 1 (HSV-1) is an infectious agent causing herpes, which affects a large population worldwide. Herpes is generally considered a benign disease whose most common form is oral herpes (commonly called "cold sores"), but it can be very serious and cause herpetic blindness and encephalitis, and even be lethal in some cases. The virus can persist throughout life in the body of its host. So far, no treatment can eliminate the virus and no vaccine has proven effective in controlling herpes infections. HSV-1 has a double-stranded DNA genome embedded in an icosahedral capsid surrounded by a lipid envelope. Thirteen viral glycoproteins are located in the envelope and are known or believed to play different roles in different stages of the viral replication cycle, including attachment, entry, assembly, and viral propagation. Among these envelope glycoproteins, glycoprotein M (gM) is the only nonessential glycoprotein but is conserved in all the herpesviridae family. Recently, the homologue of gM in Pseudorabies virus (PRV), another herpesvirus, has been implicated in the final phase of assembly (e.g. the cytoplasmic envelopment) at the trans-Golgi network (TGN) ([1]). However, it was suggested that this does not apply to HSV-1 ([2]). Moreover, unlike its TGN localization in transfected cells, HSV-1 gM localizes to the nuclear membrane and on the perinuclear virions during infection. The objective of the project presented here was to clarify the relationship of the location and function of HSV-1 gM in the context of an infection. In the results reported here, we first describe a specific and active mechanism of nuclear targeting of HSV-1 gM. In early phase of infection, gM is targeted to the nuclear membrane in a virus dependent manner. This occurs before the known reorganization of the TGN induced by the virus and before gM enters the secretory pathway. This active and specific nuclear targeting of gM seemingly does not depend on the functional interaction partners proposed in the literature. These data suggest that nuclear gM could have a new role independent of that in the final envelopment in the cytoplasm. In the second part of the work presented here, we focused iv our efforts on the role of gM in virus assembly in the late phase of infection and define an important functional domain within gM. Our results highlight the importance of the carboxyl-terminal domain of gM in the intracellular transport of gM from endoplasmic reticulum (ER) to Golgi apparatus, in the cytoplasmic envelopment of the capsids and the intercellular spread of the virus. Hence, gM ER export was completely compromised in transfected cells after deletion of its C-terminal tail. Deletion of the gM cytoplasmic tail in mutant viruses resulted in a slight reduction in viral titer and plaque size. The analysis of these mutants by electron microscopy showed an accumulation of nucleocapsids without envelope in the cytoplasm compared to wild-type virus. Interestingly, this phenotype is apparent in BHK cells but not in 143B cells, hinting that the importance of gM may be cell type specific. Finally, screening of interaction partners of C-terminal domain of gM identified by the two-hybrid system allowed us to propose several interesting candidates that may regulate the function of gM in the virus morphogenesis and propagation.

Le Virus Herpès Simplex de type 1 (HSV-1) est un agent infectieux qui cause l’herpès chez une grande proportion de la population mondiale. L’herpès est généralement considéré comme une maladie bénigne dont la forme la plus commune est l'herpès labial (communément appelé « bouton de fièvre »), mais elle peut se révéler très sérieuse et causer la cécité et l’encéphalite, voir létale dans certain cas. Le virus persiste toute la vie dans le corps de son hôte. Jusqu'à présent, aucun traitement ne peut éliminer le virus et aucun vaccin n’a été prouvé efficace pour contrôler l’infection herpétique. HSV-1 est un virus avec un génome d’ADN bicaténaire contenu dans une capside icosaèdrale entourée d’une enveloppe lipidique. Treize glycoprotéines virales se trouvent dans cette enveloppe et sont connues ou supposées jouer des rôles distincts dans différentes étapes du cycle de réplication viral, incluant l'attachement, l'entrée, l’assemblage, et la propagation des virus. La glycoprotéine M (gM) qui figure parmi ces glycoprotéines d’enveloppe, est la seule glycoprotéine non essentielle mais est conservée dans toute la famille herpesviridae. Récemment, l’homologue de gM dans le Pseudorabies virus (PRV), un autre herpesvirus, a été impliqué dans la phase finale de l’assemblage (i.e. l’enveloppement cytoplasmique) au niveau du réseau trans-Golgi (TGN) en reconnaissant spécifiquement des protéines tégumentaires et d’autres glycoprotéines d’enveloppe ([1]). Toutefois, il a été proposé que cette hypothèse ne s’applique pas pour le HSV-1 ([2]). De plus, contrairement à la localisation au TGN dans les cellules transfectées, HSV-1 gM se localise dans la membrane nucléaire et sur les virions périnucléaires durant une infection. L’objectif du projet présenté ici était d’éclaircir la relation de la localisation et la fonction de HSV-1 gM dans le contexte d’une infection. Dans les résultats rapportés ici, nous décrivons tout abord un mécanisme spécifique de ciblage nucléaire de HSV-1 gM. En phase précoce d’une infection, gM est ciblée à la membrane nucléaire d'une manière virus ii dépendante. Cela se produit avant la réorganisation du TGN normalement induite par l’infection et avant que gM n’entre dans la voie de sécrétion. Ce ciblage nucléaire actif et spécifique de gM ne semble pas dépendre des plusieurs des partenaires d’interaction proposés dans la littérature. Ces données suggèrent que la forme nucléaire de gM pourrait avoir un nouveau rôle indépendant de l’enveloppement final dans le cytoplasme. Dans la deuxième partie du travail présenté ici, nous avons concentré nos efforts sur le rôle de gM dans l’assemblage du virus en phase tardive de l’infection et en identifiant un domaine critique de gM. Nos résultats mettent en valeur l’importance du domaine carboxyl-terminal cytoplasmique de gM dans le transport de gM du réticulum endoplasmique (RE) à l’appareil de Golgi, dans l’enveloppement cytoplasmique et la propagation intercellulaire du virus. Ainsi, l’export du RE de gM a été complètement compromis dans les cellules transfectées exprimant un mutant de gM dépourvu de sa région C-terminale. La délétion la queue cytoplasmique de gM cause une réduction légère du titre viral et de la taille des plaques. L'analyse de ces mutants par microscopie électronique a démontré une accumulation des nucléocapsides sans enveloppe dans le cytoplasme par rapport aux virus de type sauvage. Étrangement, ce phénotype était apparent dans les cellules BHK mais absent dans les cellules 143B, suggérant que la fonction de gM dépende du type cellulaire. Finalement, le criblage de partenaires d’interaction du domaine C-terminal de gM identifiés par le système de double-hybride nous a permis de proposer plusieurs candidats susceptibles de réguler la fonction de gM dans la morphogénèse et la propagation de virus. / Herpes Simplex Virus type 1 (HSV-1) is an infectious agent causing herpes, which affects a large population worldwide. Herpes is generally considered a benign disease whose most common form is oral herpes (commonly called "cold sores"), but it can be very serious and cause herpetic blindness and encephalitis, and even be lethal in some cases. The virus can persist throughout life in the body of its host. So far, no treatment can eliminate the virus and no vaccine has proven effective in controlling herpes infections. HSV-1 has a double-stranded DNA genome embedded in an icosahedral capsid surrounded by a lipid envelope. Thirteen viral glycoproteins are located in the envelope and are known or believed to play different roles in different stages of the viral replication cycle, including attachment, entry, assembly, and viral propagation. Among these envelope glycoproteins, glycoprotein M (gM) is the only nonessential glycoprotein but is conserved in all the herpesviridae family. Recently, the homologue of gM in Pseudorabies virus (PRV), another herpesvirus, has been implicated in the final phase of assembly (e.g. the cytoplasmic envelopment) at the trans-Golgi network (TGN) ([1]). However, it was suggested that this does not apply to HSV-1 ([2]). Moreover, unlike its TGN localization in transfected cells, HSV-1 gM localizes to the nuclear membrane and on the perinuclear virions during infection. The objective of the project presented here was to clarify the relationship of the location and function of HSV-1 gM in the context of an infection. In the results reported here, we first describe a specific and active mechanism of nuclear targeting of HSV-1 gM. In early phase of infection, gM is targeted to the nuclear membrane in a virus dependent manner. This occurs before the known reorganization of the TGN induced by the virus and before gM enters the secretory pathway. This active and specific nuclear targeting of gM seemingly does not depend on the functional interaction partners proposed in the literature. These data suggest that nuclear gM could have a new role independent of that in the final envelopment in the cytoplasm. In the second part of the work presented here, we focused iv our efforts on the role of gM in virus assembly in the late phase of infection and define an important functional domain within gM. Our results highlight the importance of the carboxyl-terminal domain of gM in the intracellular transport of gM from endoplasmic reticulum (ER) to Golgi apparatus, in the cytoplasmic envelopment of the capsids and the intercellular spread of the virus. Hence, gM ER export was completely compromised in transfected cells after deletion of its C-terminal tail. Deletion of the gM cytoplasmic tail in mutant viruses resulted in a slight reduction in viral titer and plaque size. The analysis of these mutants by electron microscopy showed an accumulation of nucleocapsids without envelope in the cytoplasm compared to wild-type virus. Interestingly, this phenotype is apparent in BHK cells but not in 143B cells, hinting that the importance of gM may be cell type specific. Finally, screening of interaction partners of C-terminal domain of gM identified by the two-hybrid system allowed us to propose several interesting candidates that may regulate the function of gM in the virus morphogenesis and propagation.

Herpes simplex virus Typ 1 (HSV-1) ist ein humanpathogenes Virus der Familie Herpesviridae. Für eine erfolgreiche Virusreplikation besitzt HSV-1 mehrere Gene, die in den meisten infizierten Zelltypen Apoptose verhindern. Im Gegensatz dazu führt die HSV-1 Infektion eines zentralen Zelltyps des Immunsystems, den unreifen dendritischen Zellen (iDCs), zu Apoptose. Dies könnte ein Aspekt der HSV-1 Immunevasion sein. Bisher waren die Ursachen der Apoptose von HSV-1 infizierten iDCs unzureichend aufgeklärt. Es wurde jedoch gezeigt, dass das antiapoptotische zelluläre Protein c-FLIP in HSV-1 infizierten iDCs reduziert ist. In dieser Arbeit wurde die c-FLIP Menge in iDCs erstmalig mit Hilfe von RNA Interferenz erfolgreich reduziert. Dies bestätigte die Bedeutung von c-FLIP für die Lebensfähigkeit von iDCs. Folglich könnte auch die Reduktion der c-FLIP Menge nach HSV-1 Infektion iDCs für Apoptose empfindlich machen. Die HSV-1 induzierte c-FLIP Reduktion erfolgte in späten Stadien der Infektion, abhängig von der ordnungsgemäßen Expression viraler „early“ und „leaky late“ Gene. Sie fand nicht auf RNA Ebene statt und war unabhängig vom Proteasom und der Bindung an den „death inducing signaling complex“. Stattdessen wurde c-FLIP wahrscheinlich von einer viralen oder zellulären Protease abgebaut. In dieser Arbeit wurde erstmals gezeigt, dass zusätzlich zu Veränderungen im zellulären Apoptosesignalnetzwerk der Mangel an einem antiapoptotischen viralen Faktor zur Apoptose von HSV-1 infizierten iDCs beiträgt. Eine Microarray Analyse der HSV-1 Genexpression ergab, dass HSV-1 Latenz-assoziierte Transkripte (LATs) in apoptotischen iDCs signifikant geringer exprimiert waren als in nicht-apoptotischen epithelialen Zellen. LATs besitzen in Neuronen und epithelialen Zellen eine antiapoptotische Aktivität. Diese könnte den Mangel an c-FLIP kompensieren. Übereinstimmend mit dieser Hypothese induzierte eine HSV-1 LAT-Deletionsmutante mehr Apoptose in iDCs im Vergleich zum Wildtyp-Virus. / Herpes simplex virus type 1 (HSV-1) is a human pathogen which belongs to the family Herpesviridae. HSV-1 encodes several genes, which serve to efficiently prevent apoptosis in most infected cell types, thereby ensuring successful virus replication. In contrast, HSV-1 infection of one central cell type of the immune system, immature dendritic cells (iDCs), results in apoptosis. This could be one aspect of HSV-1 immunevasion. So far, the mechanisms underlying apoptosis of HSV-1 infected iDCs were poorly defined. However, it has been shown that the antiapoptotic cellular protein c-FLIP is reduced in HSV-1 infected iDCs. In this work, the amount of c-FLIP was for the first time successfully reduced in iDCs by RNA interference. This confirmed the importance of c-FLIP for viability of iDCs. Therefore, it is likely that c-FLIP reduction after HSV-1 infection also sensitizes iDCs to apoptosis. HSV-1 induced c-FLIP reduction occurred at late stages of infection and was dependent on proper expression of early and leaky late virus genes. Furthermore, it was not operative at the RNA level and was independent from the proteasome and binding to the death inducing signaling complex. Rather, c-FLIP was presumably degraded by a viral or cellular protease. In this work it was shown for the first time, that in addition to changes in the cellular apoptosis signaling network, the lack of one antiapoptotic viral factor contributes to apoptosis of HSV-1 infected iDCs. HSV-1 latency-associated transcripts (LATs) were significantly lower expressed in apoptotic iDCs compared to non-apoptotic epithelial cells, determined by microarray analysis of HSV-1 gene expression. It is known that in neurons and epithelial cells, LATs possess a potent antiapoptotic activity. This could compensate the lack of c-FLIP. Consistent with this hypothesis, a LAT deletion mutant of HSV-1 induced more apoptosis in iDCs compared to the respective wild type virus.

No description available.