Analysis of Cellular and Viral Proteins That Interact With the IE63 Protein of Herpes Simplex Virus Type 1

Bryant, Helen Elizabeth (2000) Analysis of Cellular and Viral Proteins That Interact With the IE63 Protein of Herpes Simplex Virus Type 1. PhD thesis, University of Glasgow.

Abstract

The aim of this work was to identify proteins which interact with the herpes simplex virus type 1 (HSV-1) protein IE63 in virus infected cells. This information has allowed insights as to how IE63 can exert its various activities and will help in the understanding of how transcription, RNA processing and RNA export are co-ordinated in infected and uninfected cells. HSV-1 is one of eight different herpesviruses to infect humans, the others are HSV-2, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus and human herpesviruses 6-8. Serological studies show that commonly throughout the world many individuals have been infected with one or more of these viruses. Although herpesviruses are generally not a problem in healthy individuals, in the immunocompromised they can cause hfe-threatening disease. As the immediate early (IE) protein of HSV-1, IE63 is the only HSV IE gene with homologues in all sequenced herpesviruses; studies of this essential protein, which may have similar functions throughout the Herpesviridae, are important to key questions about herpes biology and the development of effective therapies. During HSV-1 infection expression of different classes of viral genes is co- ordinately regulated and sequentially ordered. The three major classes of genes, IE, early (E) and late (L), can be separated on the basis of the kinetics of their expression and requirements for ongoing viral DNA synthesis. At the core of this regulation are three of the IE proteins, IE175, IE110 and IE63. Both IE175 and IE63 are essential for viral growth and IE110, while not essential, confers growth advantage in cell culture and is essential for establishment and reactivation from latency. IE63 is thought to repress expression of viral IE and E genes, enhance expression of viral L genes, repress expression of cellular genes and is required for viral DNA replication. IE63 regulates gene expression at the level of transcriptional and post-transcriptional control. Transcriptionally it can act on its own or with IE 175; post-transcriptionally IE63 inhibits splicing, redistributes splicing factors, increases RNA 3' processing at weak virus poly (A) sites, binds RNA and shuttles between the nucleus and the cytoplasm. Presented here are the results of co-immunoprecipitation and fusion protein pull down assays, which confirm that 1E63 interacts with four cellular proteins, namely: heterogeneous ribonucleoprotein K (hnRNP K), casein kinase 2 (CK2), p32 and splicing associated protein 145 (SAP 145). Further, hnRNP K, CK2 and either p32 or SAP 145 are complexed together only in the presence of IE63, and interaction of IE63 with p32 or SAP 145 excluded interaction with the other. Immunofluoresence demonstrated that 1E63 causes p32 to redistribute from the cytoplasm to the nucleus, and that SAP 145 and IE63 co-localise in punctate spots in the nucleus. Interestingly, when CK2 was co-immunoprecipitated with IE63 monoclonal antibody, this activity could phosphorylate other proteins in the co-immunoprecipitated complex including IE63, hnRNP K and p32. Phosphorylation of IE63 by CK2 increased the affinity of IE63 for hnRNP K. A fifth protein was present in the complex. The viral thymidine kinase (vTK) was identified by Laser Mass Map Spectroscopy, and confirmed to be associated with the IE63 complex by Western blotting of the 1E63 co-immunoprecipitates, and fusion protein pull downs. Whether IE63 interacts directly with vTK or indirectly as a consequence of its interaction with one of IE63's partners remains to be determined. How these various interactions contribute to IE63 function can be inferred from the known roles for each interacting protein; but their identification points to IE63, like a growing number of proteins involved in gene regulation, being a multifunctional protein which interacts with several proteins to carry out its functions. Interaction with transcription factors, hnRNP K and possibly p32, may account for IE63s transcriptional activity acting to enhance L gene expression and repress that of viral IE and E genes. However most of the IE63 activity is seen at the post- transcriptional level. Inhibition of splicing (via p32 and/or SAP 145), probably blocks expression of cellular genes and may also allow viral transcripts to bypass the splicing machinery before export to the cytoplasm. The redistribution of splicing factors in infected cell nuclei is most likely a reflection of the build up of viral transcripts at inactive splice sites. Export of viral mRNA serves to enhance viral gene expression and may occur via interaction with hnRNP K or p32, alternatively interaction with these proteins may block cellular mRNA export allowing viral mRNA to saturate the export pathways. IE63 almost certainly uses more than one route to facilitate export of viral mRNA. The affect of IE63 on viral DNA replication is likely to reflect its requirement for expression of DNA replication factors, but conceivably there could be a role for the IE63-TK interaction here. Multiple interactions by IE63 may be facilitated by its ability to oligomerise enabling the formation of large complexes of proteins. Moreover, the particular patterns of protein:protein interaction may be dynamic, changing at different intracellular locations or times post-infection to facilitate the various functions of IE63.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Barklie Clements
• Keywords: Virology, Molecular biology
• Date of Award: 2000
• Depositing User: Enlighten Team
• Unique ID: glathesis:2000-75547
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:30
• Last Modified: 19 Nov 2019 19:30
• URI: https://theses.gla.ac.uk/id/eprint/75547