Mechanisms of coronavirus replication: the role of cellular lipids in the generation of replication organelles
Lead Research Organisation:
University College London
Department Name: Institute of Ophthalmology
Abstract
Following entry into the host cell, coronaviruses, including SARS-CoV2, remodel cellular
membranes to form replication organelles. A characteristic feature of this membrane remodelling
is the formation of distinctive double membrane vesicles (DMVs) in which the viral RNA resides
and replicates.
DMV generation involves rewiring of host lipid metabolism. We found that expression of two
SARS-CoV-2 proteins (nsp3C and nsp4) is sufficient to induce abundant DMV formation in human
cell lines, with a corresponding upregulation of host cell lipogenic pathways that our preliminary
data suggests is essential for the formation of DMVs. Replication of other viruses has been shown
to depend on lipid transport from host cell organelles at sites of contact between the outer DMV
membrane and that of the host organelle. We have identified extensive membrane contact sites
between DMVs and a variety of host cell organelles in SARS-CoV-2 infected lung epithelial cells.
In the proposed study, we aim to generate stable inducible SARS-CoV-2 nsp3C/nsp4 cell lines to
use as a model of SARS-CoV-2 replication. Using this model, we will elucidate key pathways that
mediate lipid provision for DMV formation, by first exploring the contribution of different cellular
lipid sources. We will further establish, by expression of key candidate SARS-CoV-2 proteins and
manipulation of membrane contact site machinery, how DMV-driven interorganelle lipid transport
mechanisms are regulated, in order to identify novel targets for potential therapeutic intervention.
Finally we will assess the effect of lipogenesis/lipid transport inhibition on DMV formation and cell
survival in SARS-CoV-2 infected cells.
membranes to form replication organelles. A characteristic feature of this membrane remodelling
is the formation of distinctive double membrane vesicles (DMVs) in which the viral RNA resides
and replicates.
DMV generation involves rewiring of host lipid metabolism. We found that expression of two
SARS-CoV-2 proteins (nsp3C and nsp4) is sufficient to induce abundant DMV formation in human
cell lines, with a corresponding upregulation of host cell lipogenic pathways that our preliminary
data suggests is essential for the formation of DMVs. Replication of other viruses has been shown
to depend on lipid transport from host cell organelles at sites of contact between the outer DMV
membrane and that of the host organelle. We have identified extensive membrane contact sites
between DMVs and a variety of host cell organelles in SARS-CoV-2 infected lung epithelial cells.
In the proposed study, we aim to generate stable inducible SARS-CoV-2 nsp3C/nsp4 cell lines to
use as a model of SARS-CoV-2 replication. Using this model, we will elucidate key pathways that
mediate lipid provision for DMV formation, by first exploring the contribution of different cellular
lipid sources. We will further establish, by expression of key candidate SARS-CoV-2 proteins and
manipulation of membrane contact site machinery, how DMV-driven interorganelle lipid transport
mechanisms are regulated, in order to identify novel targets for potential therapeutic intervention.
Finally we will assess the effect of lipogenesis/lipid transport inhibition on DMV formation and cell
survival in SARS-CoV-2 infected cells.
Publications
Wong L
(2021)
Exploiting Connections for Viral Replication
in Frontiers in Cell and Developmental Biology
Wong LH
(2022)
Thank ORP9 for FFAT: With endosomal ORP10, it's fission accomplished!
in The Journal of cell biology
| Description | Through this award, we have identified an inhibitor of SARS-CoV-2 replication in human lung carcinoma cells. We are now testing the inhibitor in primary human airway cells and applying for funding to test its safety/toxicity. We are also developing a virus-free model of SARS-CoV-2 replication that can be safely used in any lab to study the formation of the replication organelle. |
| Exploitation Route | If the inhibitor is not toxic then it could potentially be developed into a therapeutic that would be predicted to be effective against replication of all coronaviruses (current and future). The replication model might be used by us and other labs to elucidate the mechanisms of replication and especially how the virus hijacks host cell machinery to protect its RNA replication from host cell defenses. |
| Sectors | Healthcare |
| Description | IoO Culture Charter |
| Geographic Reach | Local/Municipal/Regional |
| Policy Influence Type | Contribution to new or Improved professional practice |
| Title | Replication model |
| Description | We have generated cells stably expressing SARS-CoV-2 NSPs as a virus-free model of replication. |
| Type Of Material | Cell line |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | The SARS-CoV-2 replication model allows us to study the host cell factors governing the formation of the replication organelle and how we can intervene. We have found that formation of the replication organelle is dependent on upregulated host cell lipid biosynthesis. Once fully validated the model will be published and the cell lines made available on request and we also plan to deposit the necessary constructs in Addgene. |
| Description | Barclay lab |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We provide cells and inhibitors to be tested for effects on viral replication |
| Collaborator Contribution | The Barclay lab infect our cells with SARS-CoV-2 variants with or without our inhibitors and fix the cells for us to image by microscopy. They also do viral titres from infected cells to support our microscopy data. |
| Impact | We are still testing potential inhibitors of SARS-CoV-2 variants. If successful we will proceed with applications for IP and for UCL translational funding support. |
| Start Year | 2021 |
| Description | SARS-CoV-2 replication |
| Organisation | University of Cambridge |
| Department | Department of Pathology |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We wrote the grant, on with our collaborator, Dr James Edgar is a co-applicant. We designed experiments to test the potential of various treatments for inhibiting SARS-CoV-2 replication and provided the reagents. |
| Collaborator Contribution | As well as providing intellectual input into the project, James has provided us with SARS-CoV-2 infected cells ± our treatments. We have analysed these cells by IF and EM. |
| Impact | We have identified an inhibitor of SARS-CoV-2 replication. We are now optimising dose/timings on primary airway epithelial cells and plan to apply for UCL Therapeutic Accelerated Support (TAS) funding to pursue a more translational approach. |
| Start Year | 2021 |
| Description | MSc student project |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Louise Wong, the postdoc driving this research, supervised a MSc student project in the lab to generate primary human airway epithelial cells and optimise delivery of inhibitors of cholesterol biosynthesis as a potential therapeutic. The student presented her work to her classmates, enjoyed and advanced the project and is currently applying for PhDs. |
| Year(s) Of Engagement Activity | 2022 |
| Description | MSc tutorials |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | As module lead on an MSc course, i and members of my team have run tutorials that includes up to date information on our work on NPC and coronavirus replication as well as the microscopy methodologies that we use. This was incorporated into assessment papers and a student chose our lab for her research project on SARS-CoV-2 replication. |
| Year(s) Of Engagement Activity | 2021 |