Entero3D · In situ 3D structures of viral replication complexes: cryo-electron tomography of enterovirus-infected cells
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2019-01-01 → 2020-12-31
- EU contribution
- €173,857
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
In situ 3D structures of viral replication complexes: cryo-electron tomography of enterovirus-infected cells
Viruses are not autonomous, they depend on the cell they infect, to replicate their genome and form new viral particles capable of infecting neighboring cells. To this end, many viruses will drastically change a cell’s internal structure and metabolic activity within hours of infection. One group of viruses causing rapid structural alterations to cells are enteroviruses. They represent a major genus of positive-sense single-stranded RNA viruses and include human pathogens such as Poliovirus, Coxsackieviruses and Rhinoviruses. They are thus responsible for a wide variety of diseases such as poliomyelitis, myocarditis, gastroenteritis and respiratory infections. Enteroviruses reorganize the internal membranes of a cell, generating replication complexes, which are the sites of viral genome replication. Specific mechanism of how cellular membranes are rearranged into viral replication complexes and how this is connected to the viral RNA replication and packaging into new virions are unknown. In this project, I study the interior of the infected cells using cryo-electron tomography. Cryo-electron tomography provides extraordinary structural details into biological processes that were before unreachable. This will help us to determine the first high-resolution structures of viral enterovirus replication complexes in cells, providing critical insights into the genome replication of these highly medically relevant viruses, and potentially generating new concepts for virus inhibitor design.
Data: CORDIS, © European Union
Project objective
The aim of this study is to gain structural insights into the genome replication of human enteroviruses (EVs), specifically Coxsackievirus B3 which is the leading cause of viral myocarditis. EVs drastically reorganize the internal membranes of a cell within hours of infection, generating replication complexes (RCs), which are the sites of viral genome replication. Some molecular determinants of RC morphogenesis have been identified, and resin-embedding EM has indicated the drastic membrane remodeling involved in RC formation. However, such approaches fail to reveal the macromolecular structural organization of RCs in cells. To further our understanding of RC assembly and activity, I propose to use cryo-electron tomography (cryo-ET) to determine 3D structures of enteroviral RCs in infected cells. Human cells will be infected on EM grids and the recently developed cryo-focused ion beam milling technology will be used to make RCs inside infected cells accessible to structural studies using cryo-ET. The determined structures will reveal the supramolecular organization of viral proteins and RNA at EV RCs. The findings will clarify the long-standing question regarding the relation between membrane topology and the RNA synthesis machinery. Subtomogram averaging methods will be used to reveal how the viral polymerase assembles into higher-order structures on the RCs membrane, and obtain high resolution structures of these assemblies. Assemblies of purified EV polymerases will be imaged by cryo-ET and compared to structures found in cells. Additionally, the effect on the Golgi apparatus of the viral proteins thought to initiate the membrane remodelling will be studied by cryo-ET. Taken together, this project will determine the first high-resolution structures of viral RCs in cells, providing critical insights into the genome replication of the highly medically relevant EVs, and potentially generating new concepts for virus inhibitor design.
Original text from CORDIS.
Participants
- UMEA UNIVERSITET · UMEACoordinatorSweden
Links
Data: CORDIS, © European Union