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Mechanisms of cell adhesion regulation by herpes simplex virus https://doi.org/10.17863/CAM.105503 Barrow, Henry Graham, Stephen 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. University of Cambridge 2023-07-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/363430 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fd9c4fd0-4416-4cac-be32-f67e24cf0d05/download 91379238d0d149b3ab26fc4b987ecb28 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/6721ea0b-24fb-41ae-822b-224db6a19c73/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Cell adhesion Focal adhesion Herpes simplex virus Integrin-linked kinase pUL21 pUL7:pUL51 Virology