Fig 2: Flavivirus NS4A and NS4B Are Dependent on the EMC for Efficient Expression(A–D) 293T cell pools stably expressing Cas9 nuclease and an sgRNA targeting EMC6 were subsequently transiently co-transfected with constructs encoding HA-tagged DENV non-structural proteins and NS1-FLAG (A, C, and D) or GFP (B) as a transfection control.(E) ImageJ (NIH) was used to quantify band intensities for western blots of each of the DENV non-structural proteins in EMC6 knockout cells compared with wild-type from (A)–(D). Each dot represents a biological replicate. Bars represent mean ± SD. The Mann-Whitney U test was performed to assess statistical significance (*p < 0.05 and **p < 0.005 compared to NS1).(F–K) HEK293 cells were transfected with siRNAs against EMC1 or a scrambled negative control (scr). Forty-eight hours later, cells were transfected with a replication-defective full-length ZIKV cDNA (F and G) or with plasmids encoding individual S-tagged ZIKV non-structural proteins NS5 (H), NS4A (I), NS4B (J), or NS2B (K). The asterisk in (G) indicates a nonspecific background band.For (A)–(D) and (F)–(K) 24 h post-transfection, cells were lysed and proteins were separated using SDS-PAGE followed by western blotting for the indicated proteins. Each blot is representative of a minimum of two biological replicates.See also Figure S3.

Fig 3: The EMC Is Required for NS4B Biogenesis but Not for Post-translational Stability(A) HEK293T cells were transfected with scrambled or EMC1 siRNAs followed by transfection with S-tagged ZIKV NS4B or Derlinl. Twenty-four hours post-transfection, cells were pulse-labeled with [35S]Met/Cys for 20 min and chased for the indicated times. NS4B-S or Derlin1-S was affinity-precipitated and subjected to SDS-PAGE, and the radiolabeled protein was visualized using phosphorimaging. Shown is a representative image from three biological replicates.(B) The protein levels of NS4B-S and Derlin1-S at t = 0 were quantified by phosphorimaging. Data from three biological replicates are plotted as mean ± SD.(C and D) Levels of labeled NS4B-S protein (C) or Derlin1-S protein (D) in cells transfected with EMC1 siRNA (closed triangles) or negative control scrambled siRNA (open circles) were quantified at the indicated chase times and normalized to the levels at t = 0 in order to determine the degradation rate after biogenesis. Data from three biological replicates are plotted as mean ± SD. (E) Wild-type or EMC6 knockout Huh 7.5.1 cells were stably transduced to express NS4B-HA, then treated with 10 µM MG132 for 3 h to inhibit proteasomal activity. Cells were then treated with 0.02% digitonin to permeabilize the plasma membrane and release cytoplasmic proteins. The digitonin-soluble supernatant was collected, and then the digitonin-insoluble material was subjected to 1% NP40 treatment to release ER luminal proteins. The fractions were separated using SDS-PAGE and visualized by western blotting. Shown is a representative blot from two independent experiments.

Fig 4: DENV Requires the EMC for ReplicationHuh 7.5.1 cells were stably transduced with pLentiCRISPRv2 lentiviral vectors encoding Cas9 nuclease and a targeting sgRNA to knock out the indicated gene or a non-targeting (NT) sgRNA control against GFP.(A) Filled circles: two independent sgRNAs were used per gene. Cells were then infected with a luciferase reporter DENV (luc-DENV), and luciferase activity was measured 3 days post-infection as relative light units (RLU). Open circles: for EMC3, cells were transfected with either EMC3 siRNA or a scrambled negative control siRNA. Two days after transfection, cells were then infected with luc-DENV, and luciferase activity was measured 3 days post-infection.(B) Cells expressing three different sgRNAs targeting EMC6 or a NT control were transfected with in vitro transcribed RNA encoding a luciferase reporter DENV replicon. Replication was assessed by the ratio between luciferase activity at 48 versus 4 h post-transfection to control for differences in transfection and translation efficiency.(C) EMC6 knockout cells were transduced to express an sgRNA-resistant HA tagged EMC6, then infected with luc-DENV, and luciferase activity was measured 3 days post-infection. Duplicate wells were lysed for immunoblotting with the indicated antibodies.Data are plotted as relative luciferase units (RLU) as a percentage of NT. Each dot represents a biological replicate, and bars show mean ± SD relative to cells expressing a non-targeting sgRNA control. Statistical significance compared with NT was determined using the Mann-Whitney U test (*p < 0.05, **p < 0.005, and ***p < 0.0005).See also Figures S1 and S2.