Exosomes Go Viral: How Does Cytomegalovirus Impact Exosome Content and Function?

Supervisors: Michael M Nevels, Simon J Powis

Project Description:
Exosomes are small vesicles secreted from cells to participate in intercellular communication events. They contain bioactive RNA (mRNAs and miRNAs), proteins, lipids and metabolites reflective of the cell type of origin. Exosomes have roles in antigen presentation, inflammation, tumour growth, pregnancy and numerous other processes. Emerging evidence suggests that host exosome pathways are hijacked by viruses and that modified exosomes released from infected cells contribute to viral spread and immune evasion (Raab-Traub & Dittmer, 2017). However, exosomes are just beginning to be analysed for their contents and functions during viral infection.

This project will study the basic mechanisms underlying how exosomes are secreted and modified upon infection with a common and conditionally harmful, but somewhat neglected virus: cytomegalovirus (CMV).

CMV is one of eight human herpesviruses and infects most of us at some point in our lives. In most healthy adults and children, this virus causes minor if any disease symptoms. However, CMV is a major pathogen in immunocompromised hosts and the leading infectious cause of birth defects worldwide. CMV is also the biologically most complex known human virus. CMV encodes a number of proteins that target host cell pathways in an attempt to evade detection and elimination by the immune system (for an example, see Harwardt et al, 2016). However, there is essentially no information on how this virus impacts upon the release or contents of exosomes. It is highly likely that CMV subverts exosomes in as yet undiscovered ways. Indeed, our preliminary findings suggest that CMV may stimulate the secretion of exosomes to shuttle important immune effectors out of infected cells. This would help the virus to escape detection and to establish persistence.

This is a novel, interdisciplinary project employing cutting-edge technologies at a crossroads between cell biology and molecular immunology. The successful candidate will use cell lines and human primary cell models to study exosomes released during CMV infection to address four major questions:

(1) Do CMV-infected cells release varying numbers of exosomes in different cell types and at different times post infection? This question will be addressed by Nanosight nanoparticle tracking analysis, a novel method used to analyse particle size distribution in a liquid based on the principles of Brownian motion and dynamic light scattering (Zheng et al, 2013).

(2) Can exosomes secreted from CMV-infected cells at select post-infection time points be separated from virus particles based on their size (exosomes are <150 nm, herpesvirus particles are >200 nm) or their contents? Addressing this question will involve differential centrifugation (separation based on size) and exosomal marker proteins, including CD9, CD63 and CD81, for antibody bead-based purification (separation based on contents).

(3) Does CMV alter the contents of exosomes? This question will be analysed using proteomics including mass spectrometry (SWATH-MS), RNA-seq and state-of-the art bioinformatics tools to identify proteins and RNAs selectively enriched in exosomes from CMV-infected versus control cells.

The results from this project will pioneer exosome research in the context of CMV infection providing fundamental biological information that may provide leads for novel antiviral strategies.

References:

Raab-Traub N, Dittmer D (2017) Viral effects on the content and function of extracellular vesicles. Nat Rev Microbiol 15(9):559-572.

Harwardt T, Lukas S, Zenger M, Reitberger T, Danzer D, Übner T, Munday DC, Nevels M, Paulus C (2016) Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNg-like response. PLoS Pathog 12(7):e1005748.

Zheng Y, Campbell EC, Lucocq J, Riches A, Powis SJ (2013) Monitoring the Rab27 associated exosome pathway using nanoparticle tracking analysis. Exp Cell Res 319(12):1706-1713.

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