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Mechanisms of cell adhesion regulation by herpes simplex virus


Type

Thesis

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Authors

Barrow, Henry 

Abstract

Herpes simplex virus (HSV)-1 is a highly prevalent human pathogen that establishes a life-long infection. HSV-1 promotes its replication and spread by expressing multi-functional proteins that extensively remodel the host cell. Three such proteins are pUL21, pUL7 and pUL51. pUL21 is a viral phosphatase adapter. pUL7 and pUL51 form a complex that localises to juxtanuclear membranes and sites of cell-matrix adhesion termed focal adhesions. All three proteins are required for efficient virus assembly, egress and cell-to-cell spread. However, the viral or cellular binding partners required for these functions have not yet been fully identified. Furthermore, very little is known about how the interactions of pUL7:pUL51 at focal adhesions promote virus replication, spread or survival.

Biotin-proximity ligation (BioID) was used to identify novel viral and cellular protein interaction partners for pUL21 and pUL7:pUL51. The IPP complex, which consists of integrin-linked kinase (ILK), PINCH and parvin, was identified as a potential interaction partner for pUL7:pUL51 at focal adhesions. Colocalisation and a direct interaction was confirmed using immunofluorescence microscopy and biochemical approaches respectively, with binding between pUL51 and ILK identified as primarily responsible for the interaction.

Expression of pUL7:pUL51 was shown to alter focal adhesion morphology and be important for preventing infected cell rounding and detachment. Cell lines recombinantly expressing pUL7:pUL51 showed that the complex directly alters cell adhesion dynamics by likely preventing focal adhesion disassembly. pUL7:pUL51 could not localise to focal adhesions and prevent cell rounding in the absence of ILK, confirming the importance of this interaction for function. However, no large defect in cell-to-cell spread was observed in the absence of ILK, suggesting this is not a mechanism by which pUL7:pUL51 promotes virus spread between cells.

When performing the BioID experiments it was observed that fusion of the biotin ligase to pUL7 had a dominant negative effect on viral replication and cell-to-cell spread. This selective pressure was exploited to perform in vitro evolution experiments in which the virus adapted to this attenuation. Whole genome sequencing of the adapted virus population identified several HSV-1 genes that may promote virus replication and cell-to-cell spread through interactions or functional relationships with pUL7:pUL51.

Focal adhesions are highly dynamic cellular platforms that mediate bidirectional signalling between the interior of the cell and the extracellular environment. This study characterises the only known example of a virus directly manipulating focal adhesion dynamics to prevent cell detachment, identifying a direct interaction between pUL7:pUL51 and ILK. The findings provide a molecular framework to understand the regulation of focal adhesions by HSV-1, accelerating future molecular and functional studies.

Description

Date

2023-07-01

Advisors

Graham, Stephen

Keywords

Cell adhesion, Focal adhesion, Herpes simplex virus, Integrin-linked kinase, pUL21, pUL7:pUL51, Virology

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge