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

The BirAR118G biotin ligase biotinylates proteins in its close proximity that must not necessarily 166 have tight, prolonged, or direct interaction (31). Therefore, the identified RTC-proximal host 167 proteins, recorded over the entire duration of the MHV replication cycle, likely include proteins 168 that display a prolonged co-localization with the MHV RTC, proteins that may locate only 169 transiently in close proximity to the RTC, and proteins of which only a minor fraction of the 170 cellular pool may associate with the RTC. To this end, we assessed the localization of a limited 171 proteins, were not amongst the significantly enriched proteins. Our data further suggest that 185 the RTC microenvironment may be highly dynamic and likely also contains proteins that are 186 only transiently present in the microenvironment or only comprise a sub-fraction of the cellular 187 pool in close proximity to the MHV RTC. terms were highly significant (p-value <0.005) (Fig. 3a , Table S2 ). Additional analysis using 195 network analysis, using STRING, revealed at least 4 distinct clusters of interacting factors 208 within this category (Fig. 3b) . Cluster I, protein transport, comprised nuclear transport 209 receptors at nuclear pore complexes, such as importins and transportins. Interestingly, this 210 cluster also contained Sec63, which is part of the Sec61 translocon (44) and has been implicated 211 in protein translocation across ER membranes. The list of RTC-proximal factors also included 212 signal recognition particles SRP54a and SRP68 (Table S2) In order to assess the potential functional relevance of RTC-proximal factors identified in our 266 MHV-BirAR118G-nsp2-mediated proximity-dependent screen, we designed a custom siRNA 267 library individually targeting the expression of each of the 513 identified RTC-proximal host 268 proteins. siRNA-treated L929 cells were infected (MOI=0.05, n=4) with a recombinant MHV 269 vesicular transport between the ER, Golgi apparatus and endosomal compartments for the 283 establishment of replication organelles, several factors contained within these pathways 284 impaired MHV replication as exemplified by the siRNA-mediated silencing of Kif11, Snx9, 285 Dnm11, Scfd1, Ykt6, Stx5a, Clint1, Aak11, or Vapa (Fig. 4b) . Consistently, ER-associated 286 protein sorting complexes associated with the ribosome and newly synthetized proteins (Naca, 287 BTF3, SRP54a, SRP68) that were revealed in the GO enrichment analysis (Fig. 3a, table S2) , 288 also appear to be required for efficient MHV replication (Fig. 4b) . 289 Furthermore, we also observed significantly reduced MHV replication upon silencing of core 290 elements of the 26S and 20S proteasome complex (Psmd1 and Psmc2, and Psmb3, 291 respectively), suggesting a crucial role of the ubiquitin-proteasome pathway for efficient CoV 292 replication (27, 52). Indeed, this finding may provide a link to the described coronavirus RTC-293 encoded de-ubiquitination activity residing in nsp3 that has been implicated in innate immune 294 evasion (16, 17, 54). 295