Understanding the effects of the egress restriction factor Dock5 in Kaposi's sarcoma-associated herpesvirus
Lead Research Organisation:
University of Leeds
Department Name: Inst of Molecular & Cellular Biology
Abstract
The Herpesvirus family includes eight human viruses that cause life-long infections and a variety of diseases, including skin lesions, encephalitis and cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human virus associated with different malignancies, including Kaposi's sarcoma. As all Herpesviruses, KSHV replicates in the nucleus where capsid assembly takes place. Following assembly, capsids have to traverse the nuclear envelope to enter the cytoplasm in their route to exit the cell. Using state-of-the-art "omic" approaches we have recently discovered that virus infection upregulates a cellular miRNA (miR-365a-3p), which in turn reduces the expression of the cellular protein Dock5. Suppression of miR-365a-3p activity (using a miRIDIAN microRNA Hairpin Inhibitor against miR-365a-3p) and over-expression of Dock5 were found to block nuclear egress of KSHV capsids.
This PhD project aims to apply a multidisciplinary approach to characterise the role of Dock 5 in KSHV and other Herpesviruses nuclear egress. Specifically, we aim to understand if Dock5 interacts directly with KSHV capsids, or if it affects cellular components (e.g. microtubules) to prevent capsid egress. Furthermore, we aim to test if Dock5 over-expression affects viral egress of other Herpesviruses, like Herpes Simplex Virus or Cytomegalovirus. If this were to be the case, it could represent a target for novel therapeutics against Herpesviruses.
The student will apply fluorescent microscopy, cellular electron microscopy and biochemical approaches to characterise the effects of the over-expression of Dock5 and the effect of miRIDIAN microRNA against miR-365a-3p on KSHV and other Herpesviruses. The student will also study by cryo-electron microscopy the capsids accumulated when Dock5 is overexpressed. Purification of these capsids will be done by a novel nanobiopsy approach that uses a nanopipette to extract the accumulated viruses from living cells. These capsids will then be further analysed by cryo-electron microscopy using the state-of-the-art electron microscopes recently installed in Leeds
This PhD project aims to apply a multidisciplinary approach to characterise the role of Dock 5 in KSHV and other Herpesviruses nuclear egress. Specifically, we aim to understand if Dock5 interacts directly with KSHV capsids, or if it affects cellular components (e.g. microtubules) to prevent capsid egress. Furthermore, we aim to test if Dock5 over-expression affects viral egress of other Herpesviruses, like Herpes Simplex Virus or Cytomegalovirus. If this were to be the case, it could represent a target for novel therapeutics against Herpesviruses.
The student will apply fluorescent microscopy, cellular electron microscopy and biochemical approaches to characterise the effects of the over-expression of Dock5 and the effect of miRIDIAN microRNA against miR-365a-3p on KSHV and other Herpesviruses. The student will also study by cryo-electron microscopy the capsids accumulated when Dock5 is overexpressed. Purification of these capsids will be done by a novel nanobiopsy approach that uses a nanopipette to extract the accumulated viruses from living cells. These capsids will then be further analysed by cryo-electron microscopy using the state-of-the-art electron microscopes recently installed in Leeds
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| MR/N013840/1 | 01/10/2016 | 30/09/2025 | |||
| 1941921 | Studentship | MR/N013840/1 | 01/10/2017 | 31/05/2022 | Benjamin Chadwick |
| Description | MRC Flexible Funding |
| Amount | £2,500 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 03/2019 |
| End | 03/2020 |
| Description | MRC Flexible Funding |
| Amount | £350 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 05/2018 |
| End | 05/2019 |
| Description | MRC Flexible Funding |
| Amount | £2,500 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2018 |
| End | 02/2019 |
| Description | Influence of diols on Rotavirus viral factories |
| Organisation | University of Leeds |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | I have been assisting Dr Alexander Borodavka by using transmission electron microscopy to image rotavirus viroplasms. Specifically the changes size and frequency upon treatment with diols. |
| Collaborator Contribution | This is chiefly Alex's project. He has helped by supplying the virally infected cells, and conducting the original experiments through fluorescent microscopy |
| Impact | Establishment of the best time frame of infection and preliminary results. |
| Start Year | 2019 |
| Description | Tulavirus cellular ultrastructure |
| Organisation | University of Leeds |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | I have been using transmission electron microscopy to better image viral nucleoproteins structures in the cytoplasm of infected cells at different time points. |
| Collaborator Contribution | This was mainly Katherine Davies project, so she set up the experiment and did all the background work. She also provided me with the viruses and cells. |
| Impact | Identification of filamentous viral structures in the cytoplasm of cells 7 and 30 days post infection. |
| Start Year | 2019 |
| Description | Cryo-electron microscopy outreach at the Astbury Conversation |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | The Astbury conversation was a conference that had an outreach element. A mixed group of undergraduate students, postgraduate students, academics, and the general public had the opportunity to freeze flowers with liquid nitrogen. This was to demonstrate how liquid nitrogen can be used to preserve biological specimens . |
| Year(s) Of Engagement Activity | 2018 |
| Description | Cryo-electron microscopy outreach at the Be Curious event |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | The Discovery Zone is hosted by the university of Leeds, and gives the general public an opportunity to see what research is going on at the university. A mixed group of undergraduate students, postgraduate students, academics, and the general public had the opportunity to freeze flowers with liquid nitrogen. This was to demonstrate how liquid nitrogen can be used to preserve biological specimens . |
| Year(s) Of Engagement Activity | 2018 |
| Description | Microbiology Outreach 'How clean are your hands' at the Discovery Zone event. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | A number of local regional schools came to the university of Leeds to learn about a science at a variety of stalls. Our stall was educating the students on microbes through hand washing. Essentially, the students would rub a UV cream onto their hands, wash them, and then using a UV light, see how much they had washed off. |
| Year(s) Of Engagement Activity | 2018,2019 |
| Description | Microbiology Outreach 'How clean are your hands' at the Headingley Festival. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | The Headingley festival is an annual festival in Leeds. There were a variety of events, but we were the only scientific one. Our stall was educating the general pubic on microbes through hand washing. Essentially, participants would rub a UV cream onto their hands, wash them, and then using a UV light, see how much they had washed off. |
| Year(s) Of Engagement Activity | 2018 |