Fig 2: Genetic reconstitution of viral infection by expression of SBDS and SPATA5. (A) Infectivity of Huh7.5 cells treated with indicated CRISPR gRNAs and complemented with lentiviral expression of firefly luciferase (Fluc) or guide-resistant host factors infected with MOI 1 YFV-Venus for 24 h and analyzed by flow cytometry. (B) Lysates from Huh7.5 cells untreated or treated with indicated CRISPR gRNAs and complemented with lentiviral expression of Fluc or guide-resistant host factors were analyzed by Western blotting using anti-SBDS or anti-SPATA5 antibodies. Blot membranes were also probed with anti-actin antibodies (indicated) (n = 3 biological replicates). Statistical significance was determined by two-way ANOVA. KO, knockout; NonTarg or NT, nontargeting CRISPR guides; WT, wild type; ****, P < 0.0001.

Fig 3: SBDS and SPATA5 regulate ribosome biogenesis. (A) Normalized MFI of control or KO Huh7.5 cells treated with 5-ethynyl-uridine (5-EU), labeled by click chemistry, and analyzed by flow cytometry. (B) Northern blots of control or KO cell RNA labeled with ETS, ITS1, and ITS2 probes. Images of rRNA gels prior to membrane transfer are shown below blots. Band sizes of rRNA species are labeled. (C) Control nontargeting, SBDS KO, or SPATA5 KO Huh7.5 cells were stained with an anti-RSL24D1 antibody (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) and imaged by confocal microscopy. (D) Quantification of cytoplasmic localized RSL24D1 in control nontargeting, SBDS KO, or SPATA5 KO Huh7.5 cells. Quantifications were done with three separate transductions of cells except SBDS, which was done with two preparations. At least six image fields were quantified per cell line per preparation. (E) SPATA5 protein domain cartoon indicating catalytic residues targeted by site-directed mutagenesis. (F) YFV-Venus infectivity assayed by flow cytometry in control or KO cells transduced with lentivirus expressing wild-type or mutant SPATA5 (F) or Fluc control. NonTarg or NT, nontargeting CRISPR guides; ns, nonspecific; Untr, untransduced control.

Fig 4: Host factor knockout cells lack high-molecular weight polysomes. (A to C) Sucrose density gradient ribosome fractionation profiles of nontargeting control (A), host factor KO cells SBDS (B), or SPATA5 (C) overlaid with WT untreated Huh7.5 cell profiles (black). (D to H) Viral infectivity heat maps of control and KO Huh7.5 cells infected with cytoplasmic (YFV, equine arteritis virus ([EAV]) or nuclear viruses (Ad5, HSV-1, IAV) for 1 h prior to treatment without (D) or with the following inhibitors: 50 µM sodium arsenite (E), 2 µM emetine (F), 2 µM cycloheximide (G), or 10 µM anisomycin (H). Infectivity determined by flow cytometry. NonTarg or NT, nontargeting CRISPR guides; Untr, untransduced control; AU, absorbance units.

Fig 5: Virally encoded protein synthesis is reduced in SBDS and SPATA5 KO cells. (A) Cartoon of hepatitis C virus (HCV) subgenomic replicon. (B) Gaussia luciferase (Gluc) synthesis from a replication-defective (GNN) and replication-competent (WT) HCV subgenomic replicon in Huh7.5 cells. (C, D) (Left panels) Gluc synthesis from replication-defective GNN (C) or replication-competent WT (D) HCV subgenomic replicon in KO and control Huh7.5 cells. (Right panels) Area under the curve (AUC) was calculated and used to test statistical significance of each condition compared to NonTarg by one-way ANOVA (n = 4 biological replicates). NonTarg, nontargeting CRISPR guides; RLU, Renilla luciferase units; Untr, untransduced control; UTR, untranslated region; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.