Rotavirus viroplasms

Rotavirus cell infection and immunofluorescence.

MA-104 Clone 1 cells (American Type Culture Collection; ATCC:CRL-2378.1; RRID:CVCL_3846) were cultured in DMEM-RS media supplemented with 5% fetal bovine serum at 37°C and 5% CO2. Prior to infection, Rhesus rotavirus (RRV) was activated with trypsin (10 μg/ml) for 30 min at 37°C. MA104 cells grown on glass coverslips were infected with RRV at a multiplicity of infection (MOI) of 1. Cells were fixed and prepared for immunofluorescence after six hours post-infection. Using a STORM imaging buffer mounting medium (1.5% glucose oxidase + 100 mM β-mercaptoethanol), the coverslips were mounted onto the center of glass slides.

Rotavirus replication machinery imaging.

Cell infection micrographs were provided kindly by Garcés and collaborators [1]. Imaging was carried out with an Olympus IX-81 inverted microscope in TIRF mode (Olympus, cellTIRFM illuminator), with an evanescence field depth of 200 nm. The objective lens used was an Olympus UApo N 100x 1.4 NA oil-immersion, with an additional 1.6x magnification lens. Excitation of Alexa-488 and Alexa-568 was provided by 488 nm and 568 nm lasers, respectively. Using a laser-modulation protocol, previously described in [1], an EMCCD camera (iXon 897, Model No:  DU-897E-CS0-#BV; Andor) was used to acquire the images. Acquisition rate was set to 20 fps with a pixel size of 100 nm. For processing with MSSR, the algorithm parameters were set as follows: AMP = 5, PSF = 3, Order = 1, GPU parallel computing = enabled, temporal analysis = Mean (100 frames).

1. Garcés S, Y. et al. Nanoscale organization of rotavirus replication machineries. elife 8, e42906, (2019).