Investigating the mechanisms of BK Polyomavirus egress and virus-host interactions.
|dc.description.abstract||BK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that infects 70 – 90% of the world’s population and causes a lifelong, silently persistent infection. In immunocompromised individuals, BKPyV replication can result in serious pathology. Bone marrow transplant patients can develop haemorrhagic cystitis, and in kidney transplant patients BKPyV replication can provoke a nephropathy that leads to deterioration of allograft function and eventual loss of the transplanted organ. There are currently no antiviral treatments with clinical efficacy against BKPyV-associated nephropathy. The relatively small viral genome and paucity of viral proteins produced by BKPyV suggests a significant reliance on the host for viral life cycle progression. However, the lack of effective antiviral treatments indicates an incomplete understanding of such virus-host interactions. Therefore, this thesis aimed to uncover novel virus-host interactions by following up the results of a high- throughput, genome-wide loss-of-function CRISPR screen, which led to the discovery of S-adenosylmethionine transferase MAT2A as a potentially important factor in early viral life cycle progression. The results presented here reveal that deletion of MAT2A results in the reduction of infected cell number and several early viral life cycle phenotypes. The mechanism behind the influence of MAT2A in the early viral life is likely to be partly due to the effect of MAT2A on cell cycle status, previously determined to have a significant effect on viral life cycle progression. Additionally, while the life cycles of non-enveloped viruses are often assumed to require cell lysis to release progeny virions, the lack of resultant acute inflammatory responses in asymptomatically infected, immunocompetent individuals implies that BKPyV may utilize a more surreptitious method of dissemination. Indeed, there is increasing evidence to suggest that BKPyV exits the cell via non-lytic means using an unconventional secretory pathway. Thus, this thesis further investigated the non-lytic egress of BKPyV by examining the effects of knocking out cellular genes thought to be involved in bypass of the Golgi apparatus on viral release, known as GORASP1 and GORASP2. It was observed that single CRISPR-mediated knockouts of each gene both lead to a reduction in viral release, with a stronger effect observed in GORASP2 knockout cells. Lastly, the BKPyV egress pathway was further investigated by comparing changes in the plasma membrane protein composition between infected cells, mock-infected cells, and cells infected with BKPyV mutant virus Δagnoprotein, which exhibits severely impaired viral egress. This aimed to reveal more information on which specific host secretion pathways might be utilized during BKPyV egress, and additionally how such pathways might be affected by BKPyV infection. It was observed that the expression of transmembrane proteins of various subcellular compartments, including those of endoplasmic reticulum and endosome/lysosome origin, were upregulated in the plasma membrane of infected cells, compared with mock-infected cells and cells infected with Δagnoprotein virus. In addition, a reduction of interleukin receptor IL1R1 was observed in WT BKPyV-infected cells that was not dependent on agnoprotein expression. Such findings could suggest not only that specific cellular trafficking pathways are affected during viral egress, but potentially also the intentional downregulation of immunoregulatory pathways and therefore a possible immune evasion strategy. In summary, the findings of this thesis have uncovered novel virus-host interactions, shed light on putative non-lytic egress pathways employed by a non- enveloped virus, and thus offer potential new therapeutic targets for the prevention of BKPyV-associated nephropathy and allograft loss.|
|dc.description.sponsorship||Department of Pathology, Kidney Research UK, The Evelyn Trust, Medical Research Council.|
|dc.rights||All Rights Reserved|
|dc.title||Investigating the mechanisms of BK Polyomavirus egress and virus-host interactions.|
|dc.type.qualificationname||Doctor of Philosophy (PhD)|
|dc.publisher.institution||University of Cambridge|
|pubs.licence-display-name||Apollo Repository Deposit Licence Agreement|