Molecular mechanisms of Oropouche virus assembly

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

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Description We have successfully developed a robust assay for investigating the assembly mechanisms of Oropouche virus using expression of the virus envelope proteins to form virus-like particles. This enables us to conduct experiments on this aspect of the virus lifecycle much more easily and without the need for BSL3 containment. A manuscript detailing this work has now been published in Journal of Virology.

We have also conducted in depth analysis of changes to the proteins in cells caused by Oropouche virus infection from experiments conducted during a research visit to Brazil in 2022. The results are very promising and have identified several proteins that are specifically targeted by the virus. We have also identified new host factors required for the efficient production of OROV particles, including a specific member of a family of proteins called Rab GTPases, which control movement inside cells . A manuscript describing these findings is being finalized to be submitted for publication soon.
Exploitation Route Our work on Oropouche virus assembly mechanisms and the development of a VLP system will be applicable to future development of diagnostic assays for this virus as well as potential vaccine candidates to treat infections caused by this virus.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Title Research Data supporting ''Co-localisation of HA-tagged Oropouche virus glycoproteins with CHarged Multivesicular body Proteins (CHMPs)'' 
Description Immunocytochemistry of N-terminally HA-tagged glycoprotein-encoding Oropouche virus medium (M) segment co-transfected into HeLa cells with C-terminally YFP-tagged charged multivesicular body (CHMP) proteins, or co-stained with endogenous CHMP6. For all images, CHMP proteins are visible in the 488 nm channel and HA-tagged OROV glycoproteins are visible in the 568 nm channel. GM130, a marker of the Golgi apparatus ('Figure 5'), or TGN46, a marker of the trans-Golgi network ('Figure 6'), are visible in the 647 nm channel. HeLa cells were seeded on glass coverslips at a density of 5×10^4 cells per well in 24 well dishes. Cells were transfected with 250 ng of DNA (split evenly by mass between the plasmids indicated) using TransIT-LT1 (Mirus) and incubated for 24 h. Where indicated, cells were treated for 6 h prior to fixation with 1 uM monensin to disrupt protein transport from the Golgi. Cells were transferred onto ice and were washed with ice-cold PBS and fixed with cold 250 mM HEPES pH 7.5, 4% (v/v) electron microscopy-grade formaldehyde (PFA, Polysciences) for 10 min on ice before and then incubated with 20 mM HEPES pH 7.5, 4% (v/v) PFA at room temperature for a further 20 min. After washing with PBS, cells were permeabilized by incubation with 0.1% saponin in PBS for 30 min before being incubated with blocking buffer (5% [v/v] FBS, 0.1% saponin in PBS) for 30 min. Primary antibodies were diluted in blocking buffer and incubated with coverslips for 2 h. Coverslips were washed five times with blocking buffer before incubation for 1 h with the relevant secondary antibodies diluted in blocking buffer. Coverslips were washed five times with blocking buffer, three times with 0.1% saponin in PBS, three times with PBS, and finally with ultrapure water. Coverslips were mounted using Mowiol 4-88 (Merck) containing 200 nM 4',6-diamidino-2-phenylindole (DAPI) and allowed to set overnight. Cells were analysed on a Zeiss confocal laser scanning microscope (LSM)780 (Zeiss). 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
URL https://www.repository.cam.ac.uk/handle/1810/343499
 
Description Bunyavirus conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact We organized a the first International Bunyavirus research conference held at Robinson College, Cambridge, on the 27-28th of June 2022. For the conference, we received 30 abstract applications from early-career international researchers with approximately 60 participants, including representatives from the vaccines industry and CEPI.
Year(s) Of Engagement Activity 2022
URL https://www.bunya2022.path.cam.ac.uk