Determination of host cell proteins constituting the molecular microenvironment of coronavirus replicase complexes by proximity-labeling

V’kovski Philip; Gerber Markus; Kelly Jenna; Pfaender Stephanie; Ebert Nadine; Braga Lagache Sophie; Simillion Cedric; Portmann Jasmine; Stalder Hanspeter; Gaschen Véronique; Bruggmann Remy; Stoffel Michael; Heller Manfred; Dijkman Ronald; Thiel Volker

Most interestingly, this custom siRNA screen identified a crucial role of the host protein 296 synthesis apparatus that was associated with the MHV RTC as indicated by the proximity-297 dependent proteomic screen (Fig. 3a, c) . Silencing of ribosomal proteins Rpl13a and Rls24d1 298 and several subunits of the eIF3 complex resulted in greatly reduced MHV replication and 299 scored with highest significance in the siRNA screen, suggesting that proximity of the host cell 300 translation machinery to the viral RTC likely has functional importance for coronavirus 301 replication (Fig. 4b) . 302 303 Active translation near sites of viral mRNA synthesis 304 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/417907 doi: bioRxiv preprint initiation factors, we extended these results in independent assays. For this, we selected all host 306 factors assigned to the category "translation" (Fig. 3a) and assessed virus replication following 307 siRNA-mediated silencing of each factor. Measurement of luciferase activity after MHV-Gluc 308 infection confirmed initial findings obtained by screening the entire siRNA library of MHV 309 RTC-proximal factors (Fig. 4c) . Specifically for Rpl13a, and eIFs 3i, 3f, and 3e viral replication 310 was reduced to levels comparable to our controls Ceacam1a (MHV receptor) and Gbf1 (24). 311