Fig. 5: The EMC3 is required for JEV replication. | Nature Communications

Fig. 5: The EMC3 is required for JEV replication.

From: CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Fig. 5

a Alignment of the nucleic acid sequences of two clonal KO cells of EMC3 with WT cells. sgRNA targeting sites are highlighted in red. The red characters “-” indicate the deleted bases, and black “^” characters indicate the inserted bases in KO cells. PAM site is indicated in blue letters. b Western blot analysis of EMC3. c Virus plaque assays. d Absolute quantitative real-time PCR. e Immunofluorescence for detection of NS3 expressed in EMC3 KO cell lines following infection with JEV. Scale bar, 200 μm. f Western blot was performed to examine the expression of the NS3 in EMC3 KO cell lines following infection with JEV. g Real-Time Cell Analyzer assay was performed to record the cell survival curves. h, i, j Rescue assays for ectopic expression of EMC3 in KO cells. RT-qPCR assay for determination of relative mRNA level of EMC3 (h); Detection of NS3 by Immunofluorescence, the original data came from Supplementary Fig. 16a (i). RT-qPCR assay for determination of relative mRNA level of JEV C gene (j). EMC3-KO-1-rescue: Transfection of pcDNA3.1-EMC3 vector in KO cells; EMC3-KO-1-NTC: Transfection of pcDNA3.1 empty vector in KO cells. k Evaluation of the effects of EMC3 knockout on virus particle assembly by negative-staining electron microscopy. Numerous scattered virus-like particles were present in the diluted cisternae of the ER (Red arrow). The pink arrow indicates KO cells displayed dramatic changes in ER morphology after JEV infection. Scale bar, 1 µm or 100 nm. PAM, protospacer adjacent motif; JEV, Japanese encephalitis virus; Mock, non-infected cells was included as negative control; MOI, multiplicities of infection; hpi, hours post-infection; KO, knockout; WT, wild-type; DAPI, 4’, 6-diamidino-2-phenylindole; kDa, kilodalton. The experiments were repeated three times with similar results and representative results shown (k). Data are represented as means ± S.D.; n = 3 (c, d, h, i, j). *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant. P values were determined by two-sided Student’s t-test.

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