Using light and electron microscopy to understand the replication of aphthoviruses
Aphthovirus is a genus of the family Picornaviridae which includes Foot-and-mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly contagious pathogen infecting cloven-hoofed animals and is hence economically important. FMDV replication takes place in the cytoplasm and ind...
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| Format: | Thesis |
| Language: | English |
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2023
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| _version_ | 1797110483753369600 |
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| author | Lai, PYA |
| author2 | Fry, E |
| author_facet | Fry, E Lai, PYA |
| author_sort | Lai, PYA |
| collection | OXFORD |
| description | Aphthovirus is a genus of the family Picornaviridae which includes Foot-and-mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly
contagious pathogen infecting cloven-hoofed animals and is hence economically
important. FMDV replication takes place in the cytoplasm and induces massive
rearrangement of the host cell membranes to facilitate virus replication.
Rearranged membranes form structures providing the site of viral genome
replication known as the replication organelle (RO). The understanding of the RO,
viral proteins and site of virus assembly is not well established. This project applies
various microscopy approaches to investigate details of aphthovirus replication in
cells.
FMDV 3A protein is known to play a key role in viral replication machinery. We
generated recombinant viruses of FMDV with various tags fused to this protein,
subsequently allowing 3A to be detected in confocal microscopy. We developed a
split-GFP system to study the dynamics of 3A protein in vitro. We showed that 3A
signals appeared contiguous to the Golgi membrane signals suggesting that it
potentially serve as a main source of membrane associated with viral replication.
This approach was taken with the aim of facilitating the development of a
correlative light electron microscopy (CLEM) system to unravel the localisation of
virus proteins and their link to RO in cells. ERAV was used as a surrogate model to
study FMDV replication in a lower containment laboratory using cryo-electron tomography (cryo-ET). Virus particles were observed associated with membrane
structures with single membrane vesicles being more predominant than double
membrane vesicles in infected cells. By sub-tomogram averaging, we
reconstructed 3-dimensional (3D) models of intracellular ERAV full and empty
particles which were compared with structures obtained for the virus purified from
tissue culture and crystallized. Additional density was identified in the ERAV empty
particles potentially corresponding to RNA contact sites inside the capsid. |
| first_indexed | 2024-03-07T07:55:29Z |
| format | Thesis |
| id | oxford-uuid:14c553e6-bd83-4761-a7a4-d42a0beccf9e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:55:29Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:14c553e6-bd83-4761-a7a4-d42a0beccf9e2023-08-18T11:14:02ZUsing light and electron microscopy to understand the replication of aphthovirusesThesishttp://purl.org/coar/resource_type/c_db06uuid:14c553e6-bd83-4761-a7a4-d42a0beccf9eVirologyMicroscopyEnglishHyrax Deposit2023Lai, PYAFry, EStuart, DClare, DBerryman, SHawes, PTuthill, TAphthovirus is a genus of the family Picornaviridae which includes Foot-and-mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly contagious pathogen infecting cloven-hoofed animals and is hence economically important. FMDV replication takes place in the cytoplasm and induces massive rearrangement of the host cell membranes to facilitate virus replication. Rearranged membranes form structures providing the site of viral genome replication known as the replication organelle (RO). The understanding of the RO, viral proteins and site of virus assembly is not well established. This project applies various microscopy approaches to investigate details of aphthovirus replication in cells. FMDV 3A protein is known to play a key role in viral replication machinery. We generated recombinant viruses of FMDV with various tags fused to this protein, subsequently allowing 3A to be detected in confocal microscopy. We developed a split-GFP system to study the dynamics of 3A protein in vitro. We showed that 3A signals appeared contiguous to the Golgi membrane signals suggesting that it potentially serve as a main source of membrane associated with viral replication. This approach was taken with the aim of facilitating the development of a correlative light electron microscopy (CLEM) system to unravel the localisation of virus proteins and their link to RO in cells. ERAV was used as a surrogate model to study FMDV replication in a lower containment laboratory using cryo-electron tomography (cryo-ET). Virus particles were observed associated with membrane structures with single membrane vesicles being more predominant than double membrane vesicles in infected cells. By sub-tomogram averaging, we reconstructed 3-dimensional (3D) models of intracellular ERAV full and empty particles which were compared with structures obtained for the virus purified from tissue culture and crystallized. Additional density was identified in the ERAV empty particles potentially corresponding to RNA contact sites inside the capsid. |
| spellingShingle | Virology Microscopy Lai, PYA Using light and electron microscopy to understand the replication of aphthoviruses |
| title | Using light and electron microscopy to understand the replication of aphthoviruses |
| title_full | Using light and electron microscopy to understand the replication of aphthoviruses |
| title_fullStr | Using light and electron microscopy to understand the replication of aphthoviruses |
| title_full_unstemmed | Using light and electron microscopy to understand the replication of aphthoviruses |
| title_short | Using light and electron microscopy to understand the replication of aphthoviruses |
| title_sort | using light and electron microscopy to understand the replication of aphthoviruses |
| topic | Virology Microscopy |
| work_keys_str_mv | AT laipya usinglightandelectronmicroscopytounderstandthereplicationofaphthoviruses |