Characterisation of Novel Secreted Antiviral Factors to Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects a majority of the human population. Like other herpesviruses, HCMV readily establishes a latent infection that persists for the lifetime of the host and undergoes periodic lytic reactivation events. In healthy individuals, HCMV infection is typically asymptomatic, while in immunocompromised individuals, such as immunosuppressed transplant recipients, HCMV infection or reactivation of latent infection results in substantial end-organ disease and mortality. HCMV is also the most common congenital viral infection and can lead to neurological sequelae such as hearing loss and developmental delay. HCMV infection induces a multi-faceted immune response that controls but never clears the virus, due to a multitude of virally-encoded immune evasion factors. The broad immune response raised against the virus includes the action of intrinsic antiviral restriction factors, NK cells, neutralising antibodies and polyfunctional CD4+ and CD8+ T-cells. Although critical roles have been established for the antiviral cytokines IFNγ and TNFα during HCMV infection, the contribution of soluble factors to viral control remains poorly understood. In this thesis I demonstrate that the complement of secreted factors (secretomes) produced by immune cells co-cultured with HCMV-infected fibroblasts are capable of restricting HCMV in an in vitro viral dissemination assay. This activity was only partially mediated by IFNγ and TNFα and did not result from type I or type III interferon action. To determine the composition of immune cell antiviral secretomes using an unbiased quantitative approach, I developed a multiplexed proteomic method using a combination of SILAC and tandem mass tag (TMT) labelling. Application of this technology enabled detailed analysis of the secretomes produced by peripheral blood mononuclear cells (PBMC) derived from four HCMV-seropositive donors when co-cultured with HCMV-infected fibroblasts, including determination of the cellular origin of each secreted protein. This analysis identified critical components of the PBMC secretory response to HCMV, including secretion of the cytokines and chemokines IFNγ, IL-6, CXCL10 and CCL8 alongside potentially novel antiviral factors. Identification of the factors responsible for direct restriction of HCMV from this dataset proved challenging, however. Subsequent proteomic characterisation of the cellular pathways stimulated by the unidentified secreted antiviral factors implicated signalling via the NFκB pathway, providing avenues of investigation that may enable identification of these factors. Application of the novel secretomics methodology described here to isolated immune cell populations will facilitate further dissection of secreted antiviral immunity to HCMV in the future.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Potts, Martin

Advisors dc:contributor.advisor

• Wills, Mark
• Weekes, Michael

• HCMV
• Human cytomegalovirus
• Secretome
• Immunology
• Virology

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.94454

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/347039