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Research Data supporting ''Co-localisation of HA-tagged Oropouche virus glycoproteins with CHarged Multivesicular body Proteins (CHMPs)''

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Barbosa, Natalia S 
Crump, Colin M 
Graham, Stephen C 


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).


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LSM image files captured by the Zeiss LSM780 microscope can be opened using freely available image analysis tools (e.g. FiJi,


confocal microscopy, fluorescence microscopy, microscopy, virology


Biotechnology and Biological Sciences Research Council (BB/S018670/1)
Wellcome Trust (098406/Z/12/B)
This work was supported by doctoral studentship and travelling fellowship from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2016/18356-4 and 2019/02945-9) to NSB, by a FAPESP Pump Priming award (2019/02418-9) to LLPdS, by a Biotechnology and Biological Sciences Research Council (BBSRC)/FAPESP pump priming award (BB/S018670/1) to CMC, LLPdS and SCG, and by a Sir Henry Dale Fellowship co-funded by the Royal Society and the Wellcome Trust (098406/Z/12/B) to SCG.