Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/65385
Title: Structural and mechanistic studies of herpes virus proteins
Authors: Patel, Vidhi Kanaiyalal
Keywords: DRNTU::Science::Biological sciences::Biochemistry
Issue Date: 2015
Source: Patel, V. K. (2015). Structural and mechanistic studies of herpes virus proteins. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: Herpes viruses are nuclear-replicating viruses, known to cause a variety of diseases in humans ranging from painful skin and genital lesions to life-threatening cancers. They have successfully evolved to evade the immune system of humans, establishing lifelong infections. Herpes Simplex Viruses (HSV) 1 and 2 are the most prevalent amongst the eight known human herpes viruses (HHV). All herpes viruses have a characteristic life cycle, divided into latent and lytic phases. They cause recurrent infections long after the primary infection, leading to more stress to the patients. Though evolutionarily each of the HHV has diverged from each other, key survival processes are still similar. Infectious Cell Protein 27 (ICP27) from HSV is a prototype of a multifunctional regulatory protein that is highly conserved in all HHVs. It is a key regulatory protein of the HSV and has the potential to interact with an array of cellular proteins and viral intronless mRNAs. ICP27 plays an essential role in viral transcription, nuclear export of intronless mRNAs, translation of viral transcripts and virion host shut-off function. The only other well-characterized functional homolog of ICP27 is ORF57 from Kaposi’s Sarcoma-associated herpes Virus (KSHV). Other homologues, especially EB2 from Epstein-Barr Virus (EBV) and ORF69 from Human Cytomegalovirus (HCMV), are being studied currently but not yet characterized in detail. In spite of its central role in viral replication and infection, very little is known about the structure and mechanistic properties of ICP27 and its homologues. We present the first crystal structure of ICP27 at a resolution of 2.0 Å. The structure reveals the C-terminal half of ICP27 to be a bundle of α-helices along with a unique zinc-fingerlike motif. The overall fold of protein suggests it to be novel compared to protein structures available in the Protein Data Bank. We present data that confirm ICP27 exists as dimer in solution, using biophysical assays. Regulation of ICP27 by phosphorylation of the C-terminal domain has also been probed. In vitro phosphorylation assays reveal the residues that may be involved in regulation of ICP27. We also present preliminary data on EB2 protein from EBV. From sequence alignments and secondary structural analyses, we demonstrate that EB2 is not only functionally similar, but probably structurally too. Further probing will be needed to analyse if all the homologues are structurally similar in spite of divergence in sequence similarity. Structural knowledge of ICP27 and its homologues may aid in a better understanding of the mechanism by which they function. It can give a better insight to how the virus survives and replicates in host cells. Development of drugs against the herpes viral infections in humans may be guided more effectively. Herpes viral proteins have posed several challenges during trials for their expression and purification on a large scale. In this thesis, we try to outline the multi-construct approach that has been successful for human protein expression. This strategy is applied to herpes proteins and we observe that the hurdles in herpes protein expression can be circumvented to a certain extent. We present data on 94 different herpes proteins and the success we obtained in expressing otherwise difficult proteins. Few of these proteins have been successfully expressed, purified, crystallized and their structures determined to a good resolution, including ICP27. The multi-construct approach has the potential to drive viral structural biology forward and boost our knowledge in this otherwise fledgling field.
URI: https://hdl.handle.net/10356/65385
DOI: 10.32657/10356/65385
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Theses

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