Figure 4. Identification of proviral factors within the coronavirus RTC microenvironment.

(a) Impact of siRNA-silencing of RTC-proximal cellular proteins on viral replication. L929 fibroblasts were reverse-transfected with siRNAs (10 nM) for 48 hr before being infected with MHV-Gluc (MOI = 0.05, n = 4). Replication was assessed by virus-mediated Gaussia luciferase expression at 15 h.p.i. and was normalized to levels of viral replication in cells targeted by scrambled siRNA controls. Target proteins to the left of the dashed line represent RTC-proximal factors whose silencing decreased viral replication. (b) Bubble plot illustrating host proteins that significantly impact MHV replication. Bubble size is proportional to the level of viral replication impairment. Colors correspond to the functional categories highlighted in Figure 3. Light grey bubbles (below the dashed line) represent host proteins that did not significantly impact MHV replication (p-value > 0.05). (c, d, e, f) Silencing of RTC-proximal components of the cellular translation machinery. Upon 48 hr siRNA silencing of factors assigned to the category ‘translation’ (Figure 3), L929 fibroblasts were infected with MHV-Gluc (MOI = 0.05, n = 3). Luciferase activity (c), cell-associated viral RNA levels (d) and viral titers (e) were assessed at 12 h.p.i.. (f) Western blot quantification of total cellular translation following silencing of a subset of the host translation apparatus. Upon 48 hr siRNA-silencing, L929 fibroblasts were pulsed with 3 µM puromycin for 60 min. Control cells were treated, prior to puromycin incubation, with 355 µM cycloheximide and 208 µM Emetin for 30 min to block protein synthesis. Cell lysates were separated by SDS-PAGE and Western blots were probed using anti-puromycin antibodies to assess puromycin incorporation into polypeptides and normalized to actin levels. Error bars represent the mean ± standard deviation, where * is p ≤ 0.05, ** is p ≤ 0.005, *** is p ≤ 0.0005 and **** is p < 0.0001.

Figure 4—figure supplement 1. (a) siRNA controls contained in each 96-well plate during siRNA-silencing of the RTC-proximal library.

Controls included the established factors such as MHV entry receptor (Ceacam1a), Gbf1, Arf1. Arfgap2 was found to moderately affect MHV replication during pilot experiments and was included to cover the entire inhibitory range. (b) Cell viability following 48 hr siRNA-silencing of components of the cellular translation machinery. (c) Expression levels of Rpl13a, eIF3E, eIF3I, eIF3F, eIF4G1, eIF4G2, eIF2ak3, Rsl24d1 following siRNA knockdown compared to expression levels in cells treated with non-targetting siRNA. (d) Visual inspection of L929 treated with siRNA targetting eIF3E, eIF3I, eIF3F, Rrbp1, Rpl13a, non-targetting siRNA (scramble). Note that RNA silencing (b) and translation activity (c) in Rpl13a-silenced cells could not be assessed, likely due to cytotoxicity observed by visual inspection of cells. (e) Western blot and western blot analysis of total cellular translation. Upon 48 hr siRNA-silencing, L929 fibroblasts were pulsed with 3 µM puromycin for 60 min. Control cells were treated, prior to puromycin incubation, with 355 µM cycloheximide and 208 µM Emetin for 30 min to block protein synthesis. Western blots were probed using anti-puromycin antibodies to assess puromycin incorporation into polypeptides and normalized to actin levels. Error bars represent the mean ±standard deviation of three independent experiments, where * is ** is p ≤ 0.005.