Promyelocytic Leukaemia proteins (PML), the core component of PML nuclear bodies (NB), are implicated in many critical cellular functions. One of those is confronting virus infection: DNA and RNA viruses target PML-NB and interact with PML proteins and this is thought to facilitate efficient replication by interfering with their antiviral functions. One of those viruses is human adenovirus type 5 (Ad5). Its early E4Orf3 protein interacts specifically with PML isoform II (PML-II), disrupting PML-NB into track-like structures to inhibit PML- antiviral responses. PML-II has been shown to be a positive regulator of type I interferon responses, as are typically induced by virus infection, through regulating the transcription factors that control this innate immune response. Given this role of PML-II and its interaction with Ad5 E4Orf3, the first question that addressed here is how does PML-II affect the progress of Ad5 infection under normal conditions? PML-NBs are also involved in cellular stress responses, being disrupted by heat shock and other stresses. Thermo-tolerance due to mild heat conditioning protects cells from more aggressive stresses such as higher temperatures through triggering the heat shock response, which helps in re-folding the affected cellular proteins. Interferon and other cellular cytokines are also induced in response to heat shock, suggesting a mechanism whereby heat stress might affect infection, possibly dependent on PML-NBs. The second question addressed here was whether cells became more resistant to infection by pre- stressing them and if so would PML-II have a role in that resistance? To address these questions, PML-II-depleted cells were first engineered by lentiviral vector delivery of specific shRNA. Physical and functional knock-down was confirmed by measuring mRNA levels for PML-II and for genes normally induced by NF-kB activation. The biology of Ad5 infection in these cells was then assessed in comparison with control cells. Ad5 gene expression displayed substantial increases with the transient or permanent depletion of PML-II in HeLa and MRC5 cells. This effect was particularly marked for late gene expression; hexon mRNA and all other late proteins showed substantial increase in PML-II depleted cells compared to several distinct control cell lines. This increase reached up to 100% with PML-II removal, quantified by flow cytometry. Virus yield also showed a 3-fold increase in PML-II depleted cells. This effect was only partly due to the impaired interferon pathway in these cells, which would be expected to augment Ad5 gene expression. The more significant factor in that increase was the overexpression of HSP70 chaperone, a specific member of the heat shock protein family, in PML-II depleted cells. siRNA-mediated HSP70 reduction caused a drastic decrease in Ad5 gene expression in PML-II Kd cells to more or less the same level seen in control cells . This effect was not NF-kB-dependent but HSP70 depletion did increase expression of interferon response genes such as ISG56. Pre-exposure of cells to mild heat stress made them significantly more resistant to Ad5 infection: such treatment reduced viral gene expression, in particular hexon and other late mRNA and protein expression, in HeLa cells and MRC5 cells. Ad5 genome replication was also reduced in pre-stressed cells compared to the control conditions. Consistent with previous studies of more extreme heat stress, mild heat stress affected the morphology of PML-NB. It also affected expression of PML protein and specifically showed an increase in PML-II mRNA expression after 3 hours of exposure to 40 ̊C compared to the control conditions. In light of the enhanced infection seen when PML-II was depleted, this increased expression of PML-II following heat stress might play a role in the reduced infection efficiency in such cells. Indeed, PML-II removal reversed the negative effect of the mild heat stress on Ad5 infection: such cells showed more late gene expression after heat stress than control cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:693221 |
| Date | January 2016 |
| Creators | Atwan, Zeenah |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/80877/ |
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