ALG5基因敲除HEK293细胞

Transmissible gastroenteritis virus (TGEV) represents a significant threat to global swine production. In the absence of effective antiviral therapies, control relies primarily on vaccination. To identify potential therapeutic targets, we performed a genome-wide CRISPR/Cas9 screen in porcine IPEC-J2 cells, which revealed asparagine-linked glycosylation 5 (ALG5), asparagine-linked glycosylation 6 (ALG6), neurofibromin 2 (NF2), and fucosyltransferase 8 (FUT8) as essential host factors for TGEV infection. Functional characterization demonstrated that ALG5, ALG6, and NF2 knockout impaired viral adsorption and internalization through disruption of aminopeptidase N (pAPN) transcription or N-glycosylation. Consistently, tunicamycin-mediated inhibition of N-glycosylation suppressed TGEV infection. In contrast, FUT8 knockout specifically affects viral internalization and early replication by preventing the formation of double-membrane vesicles (DMVs) but does not affect pAPN expression. This role was independent of FUT8's fucosyltransferase activity, as the enzymatic inhibitor FDW028 had no effect. Mechanistically, we found that FUT8 interacts with the TGEV nonstructural proteins NSP3 and NSP4 to facilitate DMV biogenesis. Our findings delineate distinct mechanisms by which host factors support TGEV infection and provide novel insights for the development of targeted antiviral strategies.