Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, often acquired during childhood, that currently infects more than 50% of the human population. The symptoms of infection are herpetic lesions that frequently appear throughout a host's life in response to stress in the orofacial or genital region. As a pathogen, HSV-1 replicates rapidly in epithelial cells, but it is also capable of infecting neurons where it can pursue a lytic or latent infection. Latency is a state of viral quiescence where the virus can persist indefinitely yet remain poised to reactivate. Latency is unique to herpesviruses and key to HSV's success, but the molecular mechanisms that govern this state are unclear. A virus-encoded E3-ubiquitin ligase, Infected Cell protein 0 (ICP0), is often correlated with latency establishment but is detected in opposition to the state of latency. During lytic infection, ICP0 has many biological roles but primarily catalyzes the addition of ubiquitin to target substrate, marking proteins for degradation or altering their function. This ubiquitination ability allows ICP0 to alter the intracellular environment making neurons conducive to lytic or latent HSV-1 infection. ICP0's neuron-specific targets, however, are unknown, representing a significant gap in knowledge. Through the studies presented in this dissertation, we identified some of the neuron-specific ubiquitination targets of ICP0 in neurons. We utilized primary adult sensory neurons of the dorsal root ganglia and HSV-1 viral strains KOS, wild-type virus encoding a fully functional ICP0, and HSV-1 n212, encoding a truncated ICP0 protein, to illuminate the mechanisms involved in establishing and maintaining HSV latency. By using adult primary neurons and functional HSV-1 strains with and without ICP0, we were able to show that ICP0 regulates host and viral proteins during the initial onset of neuronal infection. We also show that based on neuronal conditions set forth before HSV-1 initial infection, host proteins will influence HSV-1 viral proteins to repress viral gene expression, thereby promoting the establishment of latency. / Doctor of Philosophy / Herpes simplex virus (HSV-1) is a virus, often acquired during childhood, that more than 50% of people have. Those who are infected with HSV-1 often have cold sores that appear in response to stress on the face or on the genitals. As a virus, HSV-1 replicates around the eyes, nose, and mouth but can also infect neurons where it can continue to replicate or establish latency. Latency is when the virus is inside the neurons but is unnoticeable and can reappear in response to stress. The state of latency is unique to herpesviruses and key to the success of HSV-1, but scientists are unsure of how it works. A protein made by the virus, Infected Cell Protein 0 (ICP0), is often correlated with the state of latency but is often present when the virus is not latent. ICP0 does a lot to support HSV-1, but it primarily destroys proteins that prevent the virus from replicating. By destroying proteins that prevent HSV-1 replication, ICP0 can help the virus make more viruses. The proteins that are destroyed by ICP0 are currently unknown, which represents a significant gap in knowledge. Through the research conducted in this dissertation, we identified some of the proteins that ICP0 destroys in neurons. We utilized neurons from the dorsal root ganglia and HSV-1 viral strain KOS, which encoded a functional ICP0, and n212, which encodes a nonfunctional ICP0, to study the mechanisms used by the virus to infect neurons. By using HSV-1 viruses with and without ICP0, we were able to show what proteins ICP0 destroys during infection in neurons. We were also able to show that HSV-1's ability to establish latency is dependent on how the neurons handle the initial onset of infection. Overall, a combination of host and viral proteins coordinates the virus's ability to establish latency and persist within a host.

Made available in DSpace on 2018-06-27T18:55:57Z (GMT). No. of bitstreams: 3 Danieli Ferrari da Rosa.pdf: 3718059 bytes, checksum: bb8ef19a5af8a3faa7687e991e0d5c3d (MD5) Danieli Ferrari da Rosa.pdf.txt: 158457 bytes, checksum: b6b83ac5211b5e9ee8b9dfba9feba1d9 (MD5) Danieli Ferrari da Rosa.pdf.jpg: 3270 bytes, checksum: 1f0862e05ecb2e7b1da2056322eeeaab (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sialic acid is the generic name of carboxylated monosaccharides family with nine carbon glycoconjugated at terminal portion. These molecule family are involved in several biological processes such cell recognition processes, platelet adhesion, migration, invasion and metastatic potential, it also work as a receptor for bacteria and viruses. High concentrations of total sialic acid in the blood have been reported in different groups of patients with brain tumors, leukemia, melanoma, carcinoma and other kinds of cancers. The cleavage of sialic acid is a crucial step in virus infection influenzae, since this acid is part of the cellular receptor that the virus uses during the process of cellular internalization. The neuraminidase, an enzyme produced by the virus, cleaves the bond between sialic acid and the viral glycoproteins, allowing the entry of viruses into cells.The aim of this study was the analysis of serum sialic acid levels in murine melanoma and Herpes Simplex virus-1 (HSV-1) infection model. In the tumor model were used C57BL/6 and in the viral model BALB/c mice. Mice were injected with 2x105 B16F10 cells subcutaneously in the thigh and the tumor progression was followed each day till it became visible. The HSV-1 infection was conducted by intraperitoneally injection of with 102 PFU of virus. The sialic acid in serum samples was quantified by thiobarbituric method in spectrophotometer at 549 nm. A standard curve with commercial sialic acid was used as parameter for quantification. The results showed that in tumor model the sialic acid was increased compared with control group and have significant difference (p <0.05) in the first day after administration of cells. For the viral infection the concentration of sialic acid showed a significant difference (p <0,05) in the first day after infection when compared infected with control group. The histological analysis in thigh of mice performed 24 hours after administration of B16F10 cells were found compact groups of round or polygonal melanocytes with clear and large cytoplasm, irregular chromatin, hyperchromatic and vacuolated nuclei, eosinophilic nucleoli and atypical mitosis. / O ácido siálico é o nome genérico dado a família de monossacarídeos carboxilados com nove átomos de carbono que aparece na porção terminal de glicoconjugados. Estas moléculas estão envolvidas em vários processos biológicos, tais como, processos de reconhecimento celular, adesão plaquetária, migração, invasão, potencial metastático, sendo também um receptor para bactérias e vírus. O aumento das concentrações séricas de ácido siálico total tem sido descrito em vários grupos de pacientes que sofrem de tumores cerebrais, leucemia, melanoma, carcinoma e outros tipos de cânceres. A clivagem do ácido siálico é um passo crucial para a infecção do vírus Influenza, uma vez que este ácido é parte do receptor celular usado pelo vírus durante o processo de internalização celular. A neuraminidase, enzima produzida pelo vírus, cliva a ligação entre o ácido siálico e as glicoproteínas virais, permitindo a entrada dos vírus nas células. O objetivo desse estudo foi analisar os níveis séricos de ácido siálico em modelo de melanoma murino e modelo de infecção herpética (HSV-1). No modelo tumoral foram utilizados camundongos C57BL/6 e no modelo viral camundongos BALB/c. Os camundongos receberam 2x105 células B16F10 através da administração subcutânea na coxa e a progressão do tumor foi acompanhada todos os dias até o tumor se tornar visível. A infecção com HSV-1 foi realizada através da administração intraperitoneal de 102 PFU de vírus. O ácido siálico das amostras de soro foram quantificadas pelo método tiobarbitúrico em espectrofotômetro à 549 nm. Uma curva padrão com ácido siálico comercial foi usada como parâmetro para a quantificação. Os resultados mostraram que as concentrações de ácido siálico no modelo tumoral foram aumentadas nos animais com tumor quando comparadas ao grupo controle e houve diferença significativa (p< 0,05) no primeiro dia após a administração das células. Para o modelo de infecção viral houve diferença significativa (p< 0,05) no primeiro dia após a infecção quando comparado o grupo infectado com o controle. Na análise histológica da coxa dos camundongos realizada após 24 horas da administração de células B16F10 foram encontrados grupos compactos de melanócitos arredondados ou poligonais, com citoplasma amplo e claro, cromatina irregular, núcleos hipercromáticos e vacuolizados, nucléolos eosinofílicos e mitoses atípicas.

Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel cellular functional pathways and specific proteins altered during virus infections, specifically the secretogranin II protein that had opposite types of regulation in reoviruses and HSV and was examined for its effects on virus replication. Further studies on the novel proteomic characteristics may provide greater understanding to the complex virus-host interactome, leading to possible antiviral targets. / October 2015

No description available.

Natural killer (NK) cells are non-specific killer cells of the innate immune system that eliminate target cells based on discrimination between self and non-self. Activation is carefully regulated through integration of signals received through both activating and inhibitory receptors. During the course of a herpes simplex virus type-1 (HSV-1) infection, NK cells can influence host susceptibility to infection with severe infections occurring in individuals with genetic defects in the NK cell response. In response to HSV infection, NK cells are recruited to the inflammatory tissue where ensuing reciprocal interactions with accessory cells and proinflammatory cytokines induce NK cell activation, cytolytic activity, and cytokine production, contributing to innate immune response and ultimately influencing the adaptive immune response. The objective of this study was to elucidate the multiple roles of NK cells during the numerous steps in anti-HSV immune induction. Accordingly, we have demonstrated that NK cells are novel helpers that assist and influence an anti-HSV immune response via the secretion of cytokines that enhance HSV-specific CD8+ T cell effector function and cytokine production. Taken together, data from this study presented the critical importance of NK cells in mounting an essential and efficient anti-HSV immunity. The key findings of our study were: 1. In the absence of NK cells, dendritic cells have decreased capacity to prime HSV-specific T cells. 2. HSV infected NK cells can be directly activated via toll-like receptor (TLR) in a MyD88-dependent mechanism; however, interaction with HSV infected dendritic cells yields optimal NK cell activation and function (CD69 and IFNγ). 3. TRAIL-expressing NK cells eliminate antigen-bearing immature dermal DCs (CD11c+CD8α-DR5+), that migrate to draining lymphoid organs, to facilitate antigen transfer to lymphoid resident CD8α+ DC for T cell cross priming. 4. 'Helpless' CD8+ T cell function, generated in the absence of CD4+ T cells, can be partially restored to wild-type levels by NK cell supplementation. 5. Treatment of NK cells with anti-CD69 antibody results in a heightened NK activated state and augments the adaptive immune response, without increasing NK cell numbers. These findings may contribute to the potential revelation of avenues to manipulate NK cells for anti-viral therapies.

Le virus de l’Herpès simplex de type 1 (HSV-1) est le pathogène humain responsable des lésions herpétiques labiales, plus communément appelé « feux sauvages ». Annuellement, il est responsable de plusieurs cas d’encéphalites et d’infections de l’appareil visuel qui sont la principale cause de cécité en Amérique du Nord. Bien qu’il existe quelques traitements antiviraux, aucun vaccin ou médicament ne permet de prévenir ou de guérir les infections causées par ce virus. Aujourd’hui, les infections produites par l’HSV-1 sont présentes partout sur la planète. Récemment, une étude en protéomique effectuée sur les virus matures extracellulaires a permis d’identifier la présence d’ubiquitines libres et d’enzymes reliées à la machinerie d’ubiquitination dans le virus. De plus, le virus exploite cette machinerie au cours de l’infection. Il est connu que certaines protéines virales sont ubiquitinées durant une infection et que le virus imite même certaines enzymes d’ubiquitination. Nous avons donc entrepris des recherches afin d’identifier des protéines virales ubiquitinées qui pourraient être présentes dans les virus matures ainsi que leurs rôles potentiels. La protéine majeure de la capside, VP5, un constituant très important du virus, a été identifiée. Nos recherches nous ont permis de caractériser le type d’ubiquitination, une monoubiquitination sur les lysines K810 et/ou K1275 de VP5. Le rôle que pourrait jouer l’ubiquitination de VP5 dans le cycle de réplication virale et dans les virus matures n’est toutefois pas encore connu. / Herpes simplex virus type 1 (HSV-1) is the human pathogen responsible for herpetic lesion such as cold sores. On a yearly basis, it is responsible for many cases of encephalitis and infections of the eye that are the most common cause of blindness in North America. Antiviral treatments exist, but no vaccines or drugs are able to prevent or cure the diseases caused by this virus. Today, infections caused by HSV-1 are present all around the world. Recently a proteomics approach was used to study mature extracellular viruses. This study highlighted the presence in the virus of free ubiquitin and ubiquitin related enzymes. Furthermore, the virus exploits this machinery during the course of infection. Also, it is known that certain virally encoded proteins are ubiquitinated and that the virus mimics some ubiquitin related enzymes. Our researches focused on identifying ubiquitinated viral proteins that could be present in mature extracellular viruses and their potential roles. The major capsid protein, VP5, an important virus component, was identified. We characterised the type of ubiquitination, a monoubiquitination of lysine K810 and/or K1275 of VP5. The role that could play the ubiquitination of VP5 in the viral cell cycle and in mature virions has yet to be identified.

Le virus de l’Herpès simplex de type 1 (HSV-1) est le pathogène humain responsable des lésions herpétiques labiales, plus communément appelé « feux sauvages ». Annuellement, il est responsable de plusieurs cas d’encéphalites et d’infections de l’appareil visuel qui sont la principale cause de cécité en Amérique du Nord. Bien qu’il existe quelques traitements antiviraux, aucun vaccin ou médicament ne permet de prévenir ou de guérir les infections causées par ce virus. Aujourd’hui, les infections produites par l’HSV-1 sont présentes partout sur la planète. Récemment, une étude en protéomique effectuée sur les virus matures extracellulaires a permis d’identifier la présence d’ubiquitines libres et d’enzymes reliées à la machinerie d’ubiquitination dans le virus. De plus, le virus exploite cette machinerie au cours de l’infection. Il est connu que certaines protéines virales sont ubiquitinées durant une infection et que le virus imite même certaines enzymes d’ubiquitination. Nous avons donc entrepris des recherches afin d’identifier des protéines virales ubiquitinées qui pourraient être présentes dans les virus matures ainsi que leurs rôles potentiels. La protéine majeure de la capside, VP5, un constituant très important du virus, a été identifiée. Nos recherches nous ont permis de caractériser le type d’ubiquitination, une monoubiquitination sur les lysines K810 et/ou K1275 de VP5. Le rôle que pourrait jouer l’ubiquitination de VP5 dans le cycle de réplication virale et dans les virus matures n’est toutefois pas encore connu. / Herpes simplex virus type 1 (HSV-1) is the human pathogen responsible for herpetic lesion such as cold sores. On a yearly basis, it is responsible for many cases of encephalitis and infections of the eye that are the most common cause of blindness in North America. Antiviral treatments exist, but no vaccines or drugs are able to prevent or cure the diseases caused by this virus. Today, infections caused by HSV-1 are present all around the world. Recently a proteomics approach was used to study mature extracellular viruses. This study highlighted the presence in the virus of free ubiquitin and ubiquitin related enzymes. Furthermore, the virus exploits this machinery during the course of infection. Also, it is known that certain virally encoded proteins are ubiquitinated and that the virus mimics some ubiquitin related enzymes. Our researches focused on identifying ubiquitinated viral proteins that could be present in mature extracellular viruses and their potential roles. The major capsid protein, VP5, an important virus component, was identified. We characterised the type of ubiquitination, a monoubiquitination of lysine K810 and/or K1275 of VP5. The role that could play the ubiquitination of VP5 in the viral cell cycle and in mature virions has yet to be identified.

Huntington’s disease (HD) and Spinocerebellar ataxia (SCA) are incurable neurodegenerative diseases that affect the central nervous system. Amyloids, highly organized protein aggregates, are a hallmark for many neurodegenerative diseases. The presence and accumulation of amyloids are toxic and constitute the major cause of neuron cell death. Both genetic and environmental factors contribute to the onset and progression of these diseases. However, despite intensive research, the underlying cause remains unclear. The role of viral infection as an environmental factor in the context of neurodegenerative diseases has not received much attention. The purpose of this study is to investigate the effects of Herpes Simplex Virus 1 (HSV-1) infection on nuclear amyloid aggregation in model cell lines of HD and SCA. The research process consists mainly of laboratory work which involved the use of several molecular techniques used in the field of biotechnology. The work comprises cultivating cells, infecting cells with HSV-1, Fluorescence microscopy, Western Blot and isolation and detection of amyloids. Western Blot is used for the analysis of specific proteins associated with protein aggregation in HD and SCA. The techniques used for detecting amyloids are Dot Blot and Antibody-staining of amyloids in cells. The results from Western Blot showed that aggregates changed in the presence of the virus. This pattern is observed for both HD and SCA1 cell lines. A big effort is done in this study to optimize Dot Blot as it is method that could be applied in every lab. Normalization of samples proved to be the most challenging part with Dot Blot. No definitive conclusions can be drawn from the Dot Blot results as reproducibility and sensitivity were lacking. This work addresses some of the difficulties encountered when working with detection of amyloids especially Dot Blot. Antibody-staining of amyloids showed that amyloids were formed in the presence of virus in comparison to non-infected. To conclude, aggregates changed, and amyloids were formed in the presence of virus. These results point to the fact that HSV-1 infection could be involved in the process of nuclear amyloid aggregation. The data presented in this thesis will need further investigation and characterization to identify the precise role of viral-induced amyloid formation in HD and SCA patient cells. / Huntingtons sjukdom (HD) och Spinocerebellära ataxier (SCA) är obotliga neurodegenerativa sjukdomar som påverkar det centrala nervsystemet. Amyloid, proteinaggregat som har en viss konformation är ett kännemärke för många neurodegenerativa sjukdomar. Ackumulering av dessa amyloider är toxiskt och är den främsta orsaken till att nervceller dör. Både genetiska faktorer och miljöfaktorer bidrar till uppkomsten och progressionen av dessa sjukdomar. Trots intensiv forskning är den bakomliggande orsaken emellertid fortfarande oklar. Virusinfektion som en potentiell miljöfaktor har i detta sammanhang inte fått mycket uppmärksamhet. Syftet med denna studie är att undersöka effekterna av Herpes Simplex Virus 1 (HSV-1) infektion på amyloid aggregering i modellcellinjer av HD och SCA. Forskningsarbetet bestod i huvudsakligen av experimentellt arbete med hjälp av flera molekylära tekniker inom bioteknikområdet som cell odling, infektering av celler med HSV-1, fluorescensmikroskopi, Western Blot och isolering och detektion av amyloider. Western Blot användes for att analysera specifika proteiner associerade med protein aggregering i HD och SCA. Amyloider detekterades med Dot Blot och med antikroppar specifika för amyloider. Resultat från Western Blot visade att amyloiderna förändras i virusinfekterade celler. Detta mönster observerades i både HD and SCA1 cellinjer. En stor bemöda görs i denna studie för att optimera Dot Blot eftersom det är en metod som kan användas i alla laboratorier. Normalisering visade sig vara det svåraste med detektion av amyloider. Inga definitiva slutsatser kan dras från dessa experiment, eftersom reproducerbarhet och känslighet var bristande. Detta arbete tar upp några av de svårigheter som uppstod vid arbetande med detektion av amyloider speciellt Dot Blot. Detektion av amyloider med antikropp visade att amyloider bildades till stor utsträckning i infekterade cellinjer i jämförelse med icke-infekterade. Sammanfattningsvis, amyloider förändrades och amyloider bildades i närvaro av virus. Dessa resultat indikerar på att HSV-1 infektion skulle kunna vara involverad i processen av amyloid aggregering. De presenterade uppgifter i detta examensarbete är preliminära och behöver följas upp med ytterligare studier för att identifiera virusens exakta roll i amyloid bildning i HD och SCA patient celler.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.