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Title: Studies of viral and cellular proteins involved in herpes simplex virus type-1 egress
Author: Ahmed, Md Firoz
ISNI:       0000 0004 7653 8595
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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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.
Supervisor: Crump, Colin Sponsor: Commonwealth Scholarship Commission in the UK ; Cambridge Commonwealth ; European & International Trust ; Cambridge University
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Virology ; HSV-1 ; Herpes simplex virus 1 ; HSV-1 egress ; HSV-1 gE/gI ; gE/gI ; glycoprotin E ; glycoprotein I ; Nipsnap ; IFITM ; Myoferlin ; MYOF ; virus egress ; SILAC ; Host virus interaction ; gIG39R ; HSV-1 KOS ; CRISPR