An Investigation of the Properties and Functions of the Herpes Associated Ubiquitin-Specific Protease, HAUSP

Kathoria, Meeta (1999) An Investigation of the Properties and Functions of the Herpes Associated Ubiquitin-Specific Protease, HAUSP. PhD thesis, University of Glasgow.

Abstract

Herpes simplex virus type 1 (HSV-1) is a common human pathogen best known as the causative agent of 'cold sores' around the mouth. It initially infects cells at the periphery, however it often spreads to the sensory neurones where it establishes life-long latent infection and from which it can be reactivated periodically to cause recurrent episodes of disease. The immediate early (IE) protein Vmw110 of HSV-1 stimulates the onset of lytic infection as well as increases the efficiency of reactivation from latency. As such, it has been proposed that Vmw110 plays an important role in the balance between lytic and latent states of infection. The mechanisms by which Vmw110 functions are poorly defined. However, earlier work in which Vmw110 was shown to migrate to discrete nuclear structures called NDIO, suggested that it exerts much of its effects through interactions with cellular proteins (Everett & Maul, 1994, Gelman & Silverstein, 1985, Maul et al, 1993). Investigations searching for such interactions resulted in the identification of a novel member of the ubiquitin-specific protease (USP) family named HAUSP (herpes- associated-ubiquitin specific protease) which both strongly and specifically interacted with Vmwl 10 (Everett et al., 1997, Meredith et al, 1995, Meredith et al, 1994). Studies described herein were initiated to improve the understanding of the role of HAUSP, both within the cell and for HSV-1 infection. In particular, experiments using a model USP enzyme assay confirmed that HAUSP was an enzymatically active member of the USP family. Furthermore, the presence of specific cysteine and histidine residues were shown to be essential for this activity. Investigations into the effect of transient expression of HAUSP in eukaryotic cells were also carried out. These studies suggested firstly that levels of intracellular HAUSP may be tightly controlled and secondly that increases in HAUSP expression might be toxic for cells. They also implied localisation of HAUSP to the NDIO domains was limited by protein-protein interactions. Work was also initiated to search for cellular proteins that interact with HAUSP. This resulted in the identification of strong and specific interactions between: the N-terminal region of HAUSP with cellular proteins of approximately 100kD and 105kD; and sequences in the C-terminal half of HAUSP with a cellular protein of approximately 40kD. Immunoprecipitation analysis supported the interaction of wild type HAUSP with cellular proteins of approximately 40kD and 105kD. It was also revealed that of these cellular proteins only the approximately 40kD cellular protein (which interacted with the C-terminus of HAUSP) was a substrate for proteasome-dependent degradation. More direct investigations were also carried out to improve our understanding of the mechanics and functioning of the Vmw110/HAUSP interaction. In particular, a variety of GST 'pull-down' assays were designed and tested to define the region of HAUSP required for this interaction. Results of this work implied that regions of HAUSP spanning between residues 529-576 and 744-861, were necessary for binding to Vmw110. Immunoprecipitation analysis experiments supported the requirement of the 529-576 region. Interestingly, this region is directly downstream of one of the two active site domains of HAUSP. The effect of the presence of Vmw110 on the normal cellular activities of HAUSP was also tested. Surprisingly, Vmw110 did not appear to reduce the catalytic activity of HAUSP, or hinder HAUSP from interacting with the approximately 40kD cellular protein previously described. Therefore, the specific functions of HAUSP in uninfected cells and during HSV-1 infection are yet to be fully characterised. However, the results presented in this thesis provide a solid foundation upon which future work leading to a deeper understanding of HAUSP functioning may be based.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Duncan McGench
• Keywords: Virology
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-75383
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 20:20
• Last Modified: 19 Nov 2019 20:20
• URI: https://theses.gla.ac.uk/id/eprint/75383