FIG 2.

Knockout of mkHAVCR1 in AGMK cells prevents HAV infection. (A) Two single guide RNAs (sgRNA) in exon 2 of the mkHAVCR1 gene, which codes for the IgV binding domain, were designed to knock out the gene in AGMK cells using the CRISPR/Cas9 gene editing technology. (B) Nucleotide sequence alignment of AGMK parental (wild type [WT]) and AGMK HAVCR1 knockout (AGMK HAVCR1 KO) cells shows a 100-nucleotide deletion in exon 2 resulting in a frameshift mutation. (C) Analysis of the expression of mkHAVCR1 in AGMK HAVCR1 KO cells. mkHAVCR1 expression at the cell surface of AGMK parental (black line) and AGMK HAVCR1 KO (red line) cells was determined by flow cytometry analysis using anti-HAVCR1 mAb 1D12. Parental AGMK cells were also stained with an isotype control (filled gray curve). (D) IF analysis of HAV-infected cells. AGMK parental and AGMK HAVCR1 KO cells were infected with HAV-Bsd for 4 days, and the cells were fixed and permeabilized, stained with anti-HAV neutralizing mAbs (green fluorescence), counterstained with DAPI nuclear dye (blue fluorescence), and observed under a fluorescence microscope. Fluorescence and phase-contrast micrographs from representative fields were taken at a magnification of ×400. The experiments whose results are shown in panels D and E were repeated at least 3 times with similar results.