Figure 7. SND1 binds the essential ORF50 RNA in KSHV-infected cells and SND1 knockdown significantly impairs KSHV lytic replication.

(a) RIP-seq identifies KSHV mRNAs as high-confidence SND1 targets at 0 hr (left panel), 8 hr (middle panel) and 20 hr (right panel) post-reactivation. Red dashed line indicates a cut-off of >2 fold change RIP/input. Red arrow points ORF50. Note that lytic genes are also detected in latent cells (0 hr) due to spontaneous KSHV reactivation and the high sensitivity of deep-sequencing. As expected, significantly lower coverage for these lytic genes was detected during latency compared with lytic replication. (b) RIP for endogenous SND1, FXR1, FXR2, PSIP1, normal rabbit IgG, YTHDF1 and YTHDF3 followed by qRT-PCR detection. ORF50-1, ORF50-2, ORF50-3 and ORF50-4 indicate primers that generate an amplicon spanning the first, second, third and fourth m⁶A peaks of the second exon of ORF50 RNA, respectively. ORF50-intron generates an amplicon spanning the m⁶A peak in the ORF50 intron. ORF50 negative primers were designed at the start of the second exon of ORF50 RNA. Fold enrichment is relative to the enrichment of the non-target 18S rRNA. Values are averages, error bars present s.d. For SND1 RIPs, n = 4 independent RIPs, for FXR1, FXR2 and normal rabbit IgG n = 3 independent RIPs. For PSIP1 and YTH proteins, n = 2 independent RIPs. (c–h) Stable cell lines expressing scramble shRNA (scr. KD) or two independent shRNAs targeting SND1 were generated using TREx BCBL1-Rta cells (c–e) or BCBL1 cells (f–h). (c) Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. n = 3 independent viral reactivations. Values are averages, error bars present s.d. ***p<0.001 using an unpaired t-test. ns = not significant. (d) Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. ORF57 antibody detects both full length ORF57 protein (black arrow) and the caspase-7-cleaved form (white arrow). For SND1 detection a rabbit polyclonal antibody was used. Western blots are representative of two independent viral reactivations. (e) Volcano plot displaying viral expression of scramble KD and SND1 KD2 cells 24 hr post-reactivation analysed by RNA-seq. The green arrow highlights the quadrant containing upregulated viral ORFs in depleted cells. The red arrow highlights the quadrant containing downregulated ORFs in depleted cells. The red dashed line denotes the FDR < 1% cut-off. (f) Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. n = 3 independent viral reactivations. Values are averages, error bars present s.d. **p<0.01, ***p<0.001 using an unpaired t-test. (g) Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. Western blots are representative of two independent viral reactivations. (h) Volcano plot displaying viral expression of scramble KD and SND1 KD2 cells 24 hr post-reactivation analysed by RNA-seq. The red and purple dashed lines denote the FDR < 1% and FDR < 5% cut-offs, respectively.

Figure 7—figure supplement 1. Sequencing coverage tracks for SND1 high-confidence viral RNA targets at 20 hr post-reactivation.

Figure 7—figure supplement 2. Sequencing coverage tracks for SND1 high-confidence viral RNA targets at 20 hr post-reactivation.

Figure 7—figure supplement 3. SND1 does not bind the KSHV ORF50 promoter.

ChIP experiments were carried out with latent and lytic (24 hr post-reactivation) BCBL1 cells. 2 µg of either RNAPII (positive control antibody), SND1 antibody or normal rabbit IgG (negative control antibody) were used per immunoprecipitation. The promoter of GAPDH was used as a positive control region to evaluate RNAPII binding. n = 3 independent viral reactivations followed by three independent chromatin immunoprecipitations. Values are averages, error bars present s.d.

Figure 7—figure supplement 4. m⁶A methylation has a pro-viral role in the KSHV lytic life cycle.

(a, b) BCBL1 cells were transduced with either scramble or METTL3-specific shRNAs. Two days post-transduction media was replaced with puromycin-containing media (3 μg/mL). Following 5 days post-transduction cells were reactivated for 24 hr and viral RNA and protein levels analysed. (c, d) Stable cell lines expressing scramble shRNA (scr. KD) or two independent shRNAs targeting the eraser FTO were generated using BCBL1 cells that had been under puromycin selection for at least 8 days. a, c) Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. n = 3 independent viral reactivations. Values are averages, error bars present s.d. *p<0.05, **p<0.01, ***p<0.001 using an unpaired t-test. ns = not significant. (b, d) Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. ORF57 antibody detects both full length ORF57 protein (black arrow) and the caspase-7-cleaved form (white arrow). Western blots are representative of two independent viral reactivations.

Figure 7—figure supplement 5. Depletion of YTH readers impairs KSHV lytic replication.

(a, b) Stable cell lines expressing scramble shRNA (scr.KD) or two independent shRNAs targeting the reader YTHDF1 were generated using BCBL1 cells. (c, d) BCBL1 cells were transduced with either scramble or YTHDF2-specific shRNAs. Following 5 days post-transduction cells were reactivated for 24 hr and viral RNA and protein levels analysed. Note that YTHDF2 protein was noticeably reduced following viral reactivation in scramble KD cells compared with latent scramble KD cells. (e, f) Stable cell lines expressing scramble shRNA (scr. KD) or two independent shRNAs targeting the reader YTHDF3 were generated using BCBL1 cells. a, c, e Cellular and viral RNA levels were measured by qRT-PCR 24 hr post-reactivation. For YTHDF1 and YTHDF2 knockdowns n = 3 independent viral reactivations. For YTHDF3 knockdown n = 4 independent viral reactivations. Values are averages, error bars present s.d. *p<0.05, **p<0.01, ***p<0.001 using an unpaired t-test. ns = not significant. b, d, f Immunoblot analysis of protein lysates from latent or 24 hr reactivated cells. ORF57 antibody detects both full length ORF57 protein (black arrow) and the caspase-7-cleaved form (white arrow). Western blots are representative of two independent viral reactivations.