Proteomic Analysis of Merkel Cell Polyomavirus

Triolo, Gianluca (2020). Proteomic Analysis of Merkel Cell Polyomavirus. MPhil thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00010ea7

Abstract

Over the past 8 years, the discovery of 11 new human polyomaviruses (HPyVs) has revived interest in this DNA tumor virus family. Although HPyV infection is widespread and largely asymptomatic, one of these HPyVs, Merkel cell polyomavirus (MCPyV), is a human tumor virus. JC virus (JCPyV), BK virus (BKPyV), HPyV7, and trichodysplasia-spinulosa virus (TSPyV) can cause non-neoplastic diseases in the setting of immunosuppression. Probably, one of the most common themes among the oncogenic viruses rests in the ability of one or more of the viral proteins to deregulate pathways involved in the control of cell proliferation.

The focus of this work is on MCPyV, which is a human pathogen. In addition to its importance in human health, there is growing interest in adapting MCPyV for drug delivery and other biotechnology applications, several viral coat proteins can spontaneously assemble into capsids in vitro with morphologies identical to the native virions and virion assembly is a powerful model system for studying protein complex formation. The protein capsid of the virion is a non-covalent association of protein subunits that is responsible for an array of functions, including cell attachment, cell entry, and DNA release. Even for the best studied family members, the mechanism of assembly is still poorly understood.

Studies on the proteins of MCPyV provide information about the composition of the virus, as well as individual virus-virus protein and virus-host protein interactions. Mass spectrometry offers a unique perspective on the properties of viruses. Its broad application to viral structure provides unique insights into many biological processes, including viral–antibody binding, protein–protein interactions and protein dynamics. Electrospray ionization (ESI) mass spectrometry is a powerful approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry. Mass spectrometry of viral proteins is now routine and since viruses are typically well characterized, in that the capsid protein and genome sequences are known, identifying a virus based on the mass of the protein or based on enzymatic digestion is relatively straightforward.

This thesis describes the use of mass spectrometry for the identification of proteins associated with the MCPy virion and for the analysis of the post-translational modifications of the capsid proteins, such as ubiquitinylation, phosphorylation and acetylation.

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