Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories

Guido Papa; Luca Venditti; Francesca Arnoldi; Elisabeth M. Schraner; Christiaan Potgieter; Alexander Borodavka; Catherine Eichwald; Oscar R. Burrone

Cell nuclei were stained with ProLongâ„¢ Diamond Antifade Mountant with 4',6-306 diamidino-2-phenylindole (DAPI, Thermo Scientific). Samples were imaged using 307 a confocal setup (Zeiss Airyscan equipped with a 63x, NA=1.3 objective), and the 308 images were processed using ZEN lite software. 309 11 nuclease-free Dulbecco's phosphate saline buffer (DPBS) for 10 min at room 313 temperature. Samples were washed twice with DPBS, and then permeabilized 314 with 70% (v/v) ethanol in RNAse-free water at +4 o C for at least 1 hour prior to 315 hybridization. Permeabilized samples were re-hydrated for 5 min in a pre-316 hybridization buffer (300 mM NaCl, 30 mM trisodium citrate, pH 7.0 in nuclease-317 free water, 10 % v/v formamide, supplemented with 2 mM vanadyl ribonucleoside 318 complex). Re-hydrated samples were hybridized with 62.5 nM of an equimolar 319 mixture of Cy3-labelled DNA probes designed to target the coding region of the 320 gene segment 6 of simian rotavirus A/SA11 (Genbank Acc. AY187029.1) using 321 Here we provide direct demonstration of the role of NSP5 in RV replication, using 408 an NSP5 knock out rRV (termed rRV-NSP5/KO) generated by reverse genetics. 409