Fig. 6: The CALR protein of the endoplasmic reticulum lumen is required for JEV replication. | Nature Communications

Fig. 6: The CALR protein of the endoplasmic reticulum lumen is required for JEV replication.

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

Fig. 6

a Alignment of the nucleic acid sequences of KO cells of CALR with WT cells. sgRNA targeting sites are highlighted in red. The black “^” characters indicate the inserted bases in KO cells. PAM site is indicated in blue letters. b Western blot analysis of CALR in KO and WT cells. c Virus plaque assays. d Absolute quantitative real-time PCR. e Immunofluorescence for detection of NS3 expressed in CALR KO cell line following infection with JEV. Scale bar, 200 μm. f Real-Time Cell Analyzer assay was performed to record the cell survival curves. g, h, i Rescue assays for ectopic expression of CALR in KO cells. RT-qPCR assay for determination of relative mRNA level of CALR (g); Detection of NS3 by Immunofluorescence, the original data came from Supplementary Fig. 16b (h). RT-qPCR assay for determination of relative mRNA level of JEV C gene for restoration of CALR expression in KO cells following infection with JEV (i). CALR-KO-rescue: Transfection of pcDNA3.1-CALR vector in KO cells; CALR-KO-NTC: Transfection of pcDNA3.1 empty vector in KO cells. j Evaluation of the effects of CALR knockout on virus particle assembly by negative-staining electron microscopy. Numerous scattered virus-like particles were present in the mitochondrial (Red arrow). The pink arrow indicates KO cells displaying dramatic changes in mitochondrial 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; DAPI, 4’, 6-diamidino-2-phenylindole; KO, knockout; WT, wild-type; kDa, kilodalton. The experiments were repeated three times with similar results and representative results shown (j). Data are represented as means ± S.D.; n = 3 (c, d, g, h, i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant. P values were determined by two-sided Student’s t-test. Source data are provided as a Source Data file.

Back to article page