Fig. 4: Mutagenesis of ac4C sites in PAN RNA diminishes KSHV lytic reactivation.
A A C-T mismatch (marked as red) at C614 of PAN RNA was detected in doxycycline-induced iSLK-KSHV cells with (NaBH4+) or without (NaBH4−) NaBH4 treatment, which was verified by Sanger sequencing (below). B ac4C sites in PAN RNA (586 to 625 nt) were mutated from C (marked as blue) to T (marked as black) based on the KSHV BAC16 genome, which was verified by Sanger sequencing (below). C iSLK-KSHV cells with PAN WT (WT) or PAN point mutation (PAN_Mut) in the KSHV genome were induced by doxycycline for 72 h. Then acRIP-qPCR was performed to detect the ac4C enrichment on PAN RNA. *, P < 0.05 by two-sided t-test versus the WT group. D After doxycycline induction for 12 and 48 h in iSLK-KSHV cells with PAN WT (WT) or PAN point mutation (PAN_Mut), the RFP (marker for KSHV infection) and GFP (marker for KSHV lytic replication) expressions were observed under a fluorescence microscope. Scar bars, 40 μm. E RNA decay assays with actinomycin D treatment for 0, 0.5, and 1 h were performed to examine the degradation rate of PAN RNA in iSLK-KSHV cells with PAN WT (WT) or PAN point mutation (PAN_Mut). *, P < 0.05 by two-way ANOVA versus the WT group. F By the assessment of ORF26, real-time DNA-PCR for cells shown in (C) was performed to detect intracellular and extracellular viral copy number after doxycycline stimulation for 72 h. *, P < 0.05 and **, P < 0.01 by two-sided t-test versus the WT group. G The expression of LANA, vIRF1 and vIL-6 proteins in cells shown in (C) were detected by Western blot. H The mRNA expression levels of ORF57, ORF65 and K8.1 were examined by RT-qPCR in cells shown in (C). *, P < 0.05 and **, P < 0.01 by two-sided t-test versus the WT group. Data represent mean ± SEM from n = 3 (E, H) or n = 5 (C, F) biological replicates shown as points. Source data are provided in a Source data file.
