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.

La glycoprotéine M (gM) du virus de l’herpès simplex de type 1 (VHS-1) est une protéine transmembranaire conservée parmi les Herpesviridae. C’est une protéine essentielle pour certains virus herpétiques. Cependant, gM est non essentielle quoiqu’importante pour les Alphaherpesvirinae tels que VHS-1 et VHS-2. En effet, lorsque gM est inhibée lors d’une infection au VHS-1, les titres viraux diminuent d’environ 10 fois. Lors de l’infection, gM migre tout d’abord vers les membranes nucléaires et ensuite vers le réseau trans Golgi (TGN). Il est connu que le transport de gM vers le noyau est dépendant de l’infection, puisque dans des cellules transfectées, gM s'accumule au TGN. Sachant que les interacteurs viraux connus de gM ne sont pas directement impliqués dans cette migration, une étude d’identification de nouveaux partenaires a été conduite. Cent soixante et onze protéines cellulaires potentiellement partenaires de gM ont précédemment été identifiées par BioID. Parmi celles-ci, 27 protéines ont été choisies pour validation étant donné leur implication au niveau du transport vésiculaire ou leur localisation aux membranes nucléaires. Mes travaux démontrent que l'Integral membrane protein 2B (ITM2B), une des protéines choisies pour validation, interagit et colocalise avec gM lors de l’infection. Par ailleurs, elle régule la production virale et la migration de gM vers le TGN lors d’infection. Étonnamment, cette protéine semble aussi être importante pour l’encapsidation du génome viral lors de l’infection. Finalement, nous avons montré que la production de capsides de type C, processus requérant ITM2B, serait impliqué dans la sortie nucléaire de gM. Ainsi, dans ce mémoire, nous établissons de nouvelles fonctions pour une protéine cellulaire, ITM2B, n’ayant pas d’implication auparavant décrites avec le VHS-1, durant la propagation de ce dernier. / Herpes simplex virus type 1 (HSV-1) glycoprotein M (gM) is a transmembrane protein conserved among the Herpesviridae. It is an essential protein for certain herpesviruses. However, gM is nonessential although important for HSV-1 and HSV-2. Indeed, when gM is inhibited during an HSV-1 infection, the viral titers decrease by tenfold. Upon infection, gM migrates first to nuclear membranes and then to the trans-Golgi network (TGN). It is known that the transport of gM to the nucleus is dependent on the infection since, in transfected cells, gM accumulates at the TGN. Knowing that the investigated gM viral interactors are not directly involved in this migration, a recent study identified by BioID 171 cellular proteins potentially partners of gM. Among these, 27 proteins were chosen for validation for their involvement in vesicular transport or localization at the nuclear membranes. Integral membrane protein 2B (ITM2B), one of the proteins chosen for validation, seems to be important for viral production as well as the migration of gM to the TGN during infection. Moreover, we showed that ITM2B interacts and colocalizes with gM upon infection. This protein also seems to be important for encapsidation of the viral genome. Finally, we showed that the production of C-type capsids, a process requiring ITM2B, would be involved in the nuclear exit of gM. In this memoir, we establish new functions for a cellular protein, ITM2B, having no involvement previously described with HSV-1, during the propagation of the latter.

La glycoprotéine M (gM) est une protéine virale transmembranaire qui est conservée dans la famille des Herpesviridae. Malgré son rôle non essentiel in vitro chez la plupart des virus de la sous-famille des Alphaherpesvirinae, dont l’herpès simplex de type 1 (VHS-1), gM est impliquée à plusieurs étapes de leur cycle viral et sa déplétion entraine une diminution de la production virale. Pour effectuer ses diverses fonctions, gM est ciblée dynamiquement à plusieurs compartiments cellulaires au cours de l’infection, dont le noyau, le réseau trans-Golgi et la membrane plasmique. Chez le VHS-1, gM est la première glycoprotéine détectée aux membranes nucléaires, et ce, dès 4 heures après le début de l’infection. Des expériences effectuées précédemment dans notre laboratoire ont démontré que la localisation de gM au noyau à 4hpi est un processus actif, viral-dépendant et spécifique qui succède sa traduction au réticulum endoplasmique. Or, sa fonction au noyau n’est toujours pas élucidée, ni le mécanisme lui permettant d’atteindre ce compartiment tôt durant l’infection. D’ailleurs, aucun des partenaires d’interaction connus de gM n’a été identifié comme participant à ce ciblage, soulevant des questions quant au mécanisme utilisé par la glycoprotéine virale pour atteindre le noyau. Notre hypothèse est que gM emprunte le transport nucléocytoplasmique de la cellule pour être activement ciblée à la membrane nucléaire interne par l’intermédiaire des importines. Afin d’étudier le rôle des importines dans la localisation de gM tôt dans l’infection, chaque importine a été déplétée par ARN interférent dans des cellules 143B. À la suite d’une infection de 4h, la localisation de gM a été déterminée par microscopie confocale suivie d’analyses qualitatives en 2D et en 3D. Les résultats obtenus suggèrent que les importines ne participent pas significativement au ciblage de gM aux membranes nucléaires à cette étape de l’infection. Ces observations ouvrent la porte à d’autres mécanismes de transport qui devront être étudiés afin de mieux comprendre le ciblage de gM à ce compartiment et, éventuellement, y déterminer son ou ses rôles dans le cycle viral de l’herpès. / Glycoprotein M (gM) is a viral transmembrane protein that is conserved in the Herpesviridae family. Despite its non-essential role in vitro in most viruses of the Alphaherpesvirinae subfamily, including herpes simplex virus type 1 (HSV-1), gM is involved at several stages of their viral cycle and its depletion leads to a decrease in viral production. To perform its various functions, gM is dynamically targeted to several cellular compartments during infection, including the nucleus, the trans-Golgi Network and the plasma membrane. In HSV-1, gM is the first glycoprotein detected at nuclear membranes as early as 4 hours after the onset of infection. Previous experiments conducted in our laboratory have shown that the localization of gM to the nucleus at 4hpi is an active, viral-dependent and specific process that follows its translation at the endoplasmic reticulum. However, its function at the nucleus is still not elucidated, nor is the mechanism by which it reaches this compartment early during infection. Moreover, none of gM's known interacting partners have been identified as participants in this targeting, raising questions about the mechanism used by the viral glycoprotein. Our hypothesis is that gM takes advantage of the nucleocytoplasmic transport of the cell to be actively targeted to the inner nuclear membrane via importins. In order to study the role of the importins in the localization of gM early in the infection, each importin was depleted by interfering RNA in 143B cells. After a 4-hour infection, gM localization was determined by confocal microscopy followed by 2D and 3D qualitative analysis. The results obtained from these experiments suggest that importins do not significantly participate in the targeting of gM to nuclear membranes at 4hpi. These observations open the door to other transport mechanisms that will need to be studied in order to better understand the targeting of gM to this compartment and to eventually determine its role(s) in the herpes viral cycle.

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