Knockout of endogenous NDRG1 impairs viral lytic replication.

(A and B) NDRG1-deficient iSLK.RGB cell line was constructed through the lentiviral CRISPR/Cas9 system. The knockout efficiency of NDRG1 was confirmed by immunoblotting and Sanger sequencing. (C and D) Wild-type iSLK.RGB cells (WT) and NDRG1-deficient iSLK.RGB cells (NDRG1^-/-) were treated with doxycycline. Intracellular viral genomic DNA and extracellular virion DNA were extracted from the induced cells or cells supernatants. The KSHV genomic DNA copy numbers at indicated time points were detected by qPCR analysis (C). The culture supernatants (1ml) collected from the indicated cells at indicated post dox induction time points were incubated with HEK293T cells. At 24 h post-infection, the virus infection rate of HEK293T cells was detected by fluorescence microscopy according to RFP signal intensity (D). (E and F) NDRG1-deficient iSLK.RGB cells were infected with the indicated lentivirus, after 24 h, the cells were treated with doxycycline and the cells and supernatants were collected at indicated time points. The protein expression of RTA and NDRG1-Flag were detected by immunoblotting (E) and intracellular and extracellular KSHV genomic DNA copy numbers at indicated time points were determined by qPCR analysis (F). (G and H) NDRG1-deficient iSLK.RGB cells were infected with the indicated lentivirus, at 24 h post-infection, the cells were induced by doxycycline, then the cells and supernatants were collected at indicated time points. The protein expression of RTA and ORF44-Flag were confirmed by immunoblotting (G) and intracellular and extracellular KSHV genomic DNA copy numbers at indicated time points were detected by qPCR analysis (H). Data were shown as mean ± SD, n = 3; ns, not significant; **p < 0.01; ***p < 0.001; ****p <0.0001.