An Investigation of the Interactions of the Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 With Cellular Proteins

Meredith, Michayla Robyn (1996) An Investigation of the Interactions of the Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 With Cellular Proteins. PhD thesis, University of Glasgow.

Abstract

HSV-1 is a human pathogen which initially infects epithelial cells (usually causing 'cold sores' around the mouth) and then establishes life-long latent infection in sensory neurons. A knowledge of the mechanisms by which latent infection is established and by which reactivation occurs is likely to be a key factor in understanding the biology of the virus. The HSV-1 virion contains a double stranded linear genome of 152kD which encodes at least 77 genes. These genes are expressed in a temporally regulated cascade during lytic infection in tissue culture. The five immediate-early genes are transactivated by a component of the virion tegument (Vmw65) in a complex with cellular proteins. The expression of at least three,Vmw175, Vmw63 and Vmw110, is necessary for the efficient transactivation of early and late genes. Vmw175 is the major viral transactivator and Vmw63 is thought to act at the post-transcriptional level. The mechanism of action of Vmw110 is unclear. Vmw110 is a 775aa phosphoprotein which is localised to discrete domains in the nucleus, called ND10, at early times of infection. The protein is encoded by gene IE1 which yields one of the five HSV-1 transcripts that are spliced. Vmw110 is non- essential in tissue culture but deletion mutant viruses exhibit a cell type and multiplicity dependent defect in plaque formation as a result of inefficient induction of early and late gene expression. Whilst Vmw110 is also non-essential for infection in mouse model systems, deletion mutant viruses reactivate inefficiently from latency. In an in vitro latency system, Vmw110 is both necessary and sufficient to reactivate quiescent viral genomes. In transfection assays, Vmw110 has been shown to be a potent and promiscuous transactivator of gene expression, particularly in synergy with Vmw175, and five functional regions have been identified on the basis of mutational analysis; one of these encompasses the RING finger and another the C-terminal end of Vmw110. The C-terminal end is important for the initial interaction of Vmwll0 with ND10; this provided circumstantial evidence for Vmw110 interacting with cellular proteins via C- terminal residues. Given the potential involvement of Vmw110 in the switch between lytic and latent infection, it was considered important to pursue any potential interactions between Vmw110 and cellular proteins. To investigate the interactions of the C-terminal end of Vmw110 with cellular proteins, a series of Vmw110 C-terminal GST fusion proteins were made and used in 'pull-down' experiments with radiolabelled whole cell extracts. Vmw110 residues 594- 775 (GEXE52) were found to bind strongly and specifically to a 135kD protein from all cell types tested and to a 150kD protein from some cell types. A shorter fusion protein containing Vmw110 residues 633-775 (GEXE58) did not interact with the 135kD protein indicating that residues 594-633 of Vmwl 10 are necessary for the interaction in this system. GEXE58 bound the 150kD cellular protein whilst a fusion protein containing Vmw110 residues 680-775 (GEXR5) did not, showing that Vmw110 residues between 633 and 680 are necessary for this interaction. Immune precipitation experiments carried out by other members of the group showed that purified, intact Vmw110 bound a 135kD protein and a collaborative reciprocal depletion experiment indicated that the 135kD proteins in the two systems were likely to be the same. In order to further understand the mechanism by which Vmw110 acts during infection, it was considered important to purify the 135kD protein and attempt the cloning of a cDNA. The 135kD protein was purified in a large scale GST 'pull-down' experiment and the fusion protein bound samples containing the 135kD protein were blotted onto a PVDF membrane. The 135kD protein band was isolated and submitted for microsequence analysis by Dr. P. Matsudaira (MIT). Six peptide sequences were obtained and the information employed in the design of redundant PCR primers for use in cloning a cDNA. This approach was unsuccessful and a long probe (53 bases) was designed based on one of the longer peptides, taking into account the optimum codon usage in the human genome (Lathe rules), for use in screening a cDNA library. This approach was also unsuccessful during the course of these studies but another member of the group was successful with this method later on. Indirect evidence from another laboratory indicated that C-terminal residues of Vmw110 contain a multimerisation domain; it was possible that such a domain could include or overlap with the residues required for 135kD protein binding, so the multimeric nature of Vmwl 10 was examined more closely. The Vmw110 E52 and E58 sequences were cloned into a T7 expression system and following expression, the Vmw110 peptides (T7E52 and T7E58) were purified by ion exchange chromatography. Analysis of the purified peptides by gel filtration chromatography, glycerol gradient centrifugation and glutaraldehyde cross-linking showed that both formed multimers of higher order than dimers although their precise nature could not be resolved. These experiments clearly separated the Vmw110 residues required for 135kD binding (594- 633) from those containing an independent multimerisation domain. The significance of the Vmw110:135kD protein:protein interaction and the function of Vmw110 multimerisation during the life cycle of the virus are unknown,as yet, but undoubtably, this work has opened a new door for studying the mechanism of action of Vmw110 during HSV-1 infection.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: John Subak-Sharpe
• Keywords: Virology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75698
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 18:55
• Last Modified: 19 Nov 2019 18:55
• URI: https://theses.gla.ac.uk/id/eprint/75698