FIG 4.

ZIC2 is essential for maintaining KSHV latent infection in BCBL-1 cells. (A) BCBL-1 cells were transduced with lentivirus expressing shScrm, shZIC2_1, or shZIC2_2. Total RNA was harvested at 72 h postransduction, and ZIC2 expression was measured by RT-qPCR. (B) At 0, 24, 48, 72, and 96 h postransduction, cell viability was measured by a trypan blue exclusion assay. (C) Viral gene expression was measured by RT-qPCR at 96 h postransduction. The relative levels of mRNA were normalized to the level of GAPDH. (D) BCBL-1 cells were transduced for 72 and 96 h. Cell lysates were immunoblotted with anti-ZIC2, anti-K-Rta, anti-ORF45, anti-K-bZIP, anti-K8.1, or anti-β-actin antibody. (E) IFA with anti-K-Rta antibody (far-red fluorescence) was performed at 72 h after lentiviral transduction to detect lytic reactivation. White rectangles, the areas shown in the enlarged views on the right; white arrowheads, ZIC2-KD cells with mild induction of K-Rta expression. (F) (Left) BCBL-1 cells were transduced for 96 and 120 h; (right) BCBL-1 cells were treated with 1 mM NaB for 120 h. The culture supernatant was collected, virion DNAs were extracted, and the copy number of virion DNA was quantified by RT-qPCR.