N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

Abstract

Methylation at the N6 position of adenosine (m6A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome. However, we found that depletion of the m6A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m6A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m6A modified. In contrast, although the ORF50 mRNA was also m6A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m6A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.

Fig 1. m⁶A increases upon KSHV reactivation.

(A) Schematic of the experimental setup. iSLK.219 cells were induced with doxycycline for 5 days to induce the lytic cycle, and total RNA was collected and subjected to oligo dT selection to purify poly(A) RNA. Polyadenylated RNA was spiked with 10 μM of 5-fluorouridine and digested with nuclease P1 and alkaline phosphatase, and subjected to LC-MS/MS analysis. (B) Relative m⁶A content in iSLK.219 cells. The induced sample was normalized with respect to the uninduced sample (set to 1).

Fig 2. KSHV mRNA contains m⁶A modifications.

(A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m⁶Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m⁶A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m⁶A IP samples, aligned to the ORF50 transcript and the annotated GG(m⁶A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m⁶A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m⁶A peaks from m⁶Aseq analysis.

Fig 3. m⁶A and the reader YTHDF2 potentiate viral gene expression and virion production in iSLK.219 cells.

Cells were transfected with control scramble (scr) siRNAs or siRNAs against METTL3, YTHDF1, 2, or 3, then reactivated for 72 hr with doxycycline and sodium butyrate. (A) Knockdown efficiency was measured by western blot using antibodies for the indicated protein, with GAPDH serving as a loading control in this and all subsequent figures. (B) Viral supernatant was collected from the reactivated iSLK.219 cells and transferred to uninfected HEK293T recipient cells. 24 hr later, the recipient cells were analyzed by flow cytometry for the presence of GFP, indicating transfer of infectious virions. (C) ORF50, ORF37 and K8.1 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs. Data are from 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values <0.05 (*), ≤ 0.01 (**), and ≤ 0.001 (***). (D) Expression of the viral ORF50 and ORF59 proteins in cells treated with the indicated siRNAs was measured by western blot 72 hr post-reactivation. (E) Unreactivated iSLK.219 cells containing latent virus were treated with control scramble (scr) siRNAs or siRNAs targeting METTL3, YTHDF1, YTHDF2, or YTHDF3. The cells were then reactivated with dox and sodium butyrate for 48 hr and lytic reactivation was monitored by expression of the lytic promoter-driven red fluorescent protein. (F) Protein was harvested from the above described cells and subjected to western blot for ORF50 and the control GAPDH protein at 24 hr post-reactivation. (G-H) Uninfected iSLK.puro cells expressing DOX-inducible RTA were transfected with the indicated siRNAs for 48 hr, then treated with dox for 24 hr to induce ORF50 expression. Knockdown efficiency (G) and ORF50 protein levels (H) were measured by western blot using antibodies for the indicated protein, with GAPDH serving as a loading control.

Fig 4. Depletion of the m⁶A writer and readers does not impact ORF50 nascent transcription in iSLK.219 cells.

(A) Schematic of the experimental setup for measuring nascent RNA synthesis. Cells were transfected with the indicated siRNAs for 48 hr then reactivated for 24 hr with dox. 4sU was added for 30 minutes, whereupon 4sU-labeled RNA was isolated using biotin/streptavidin affinity purification, reverse transcribed, and analyzed by RT-qPCR using primers specific to ORF50 or ORF37. (B-D) Levels of 4sU-labeled total ORF50 (B), ORF37 (C), and ORF50 transcribed from the viral genome (virus-derived) (D) determined as described above. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values <0.05 (*), ≤ 0.01 (**), and ≤ 0.001 (***).

Fig 5. METTL3 and YTHDF2 are important for KSHV lytic replication in iSLK.BAC16 cells.

Cells were transfected with control scramble (scr) siRNAs or siRNAs against METTL3 or YTHDF2, then reactivated for 24 hr with doxycycline and sodium butyrate. (A) Knockdown efficiency was measured by western blot using antibodies for the indicated protein. (B) Viral supernatant was collected from the reactivated iSLK.BAC16 cells 72 hr post-reactivation and transferred to uninfected HEK293T recipient cells. 24 hr later, the recipient cells were analyzed by flow cytometry for the presence of GFP, indicating transfer of infectious virions. (C) Quantification of supernatant transfer results from four independent experiments. (D) ORF50, ORF37, and K8.1 gene expression 24 hr post-reactivation was analyzed by RT-qPCR from cells treated with the indicated siRNAs. Data for ORF50 are from five independent experiments, while ORF37 and K8.1 data are from four independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples in panels C-D. Significance is shown for P values ≤ 0.01 (**) and ≤ 0.001 (***). (E) Western blots showing expression of the viral ORF50 and ORF59 proteins at 24 hr post reactivation of iSLK.BAC16 cells treated with the indicated siRNAs.

Fig 6. Increased viral gene expression upon m⁶A writer and reader depletion in TREX-BCBL-1 cells.

(A) TREX-BCBL-1 cells were reactivated with dox for 72 hr, then total RNA was isolated and subjected to m⁶A RIP, followed by RT-qPCR for analysis of KSHV ORF50, GAPDH, and DICER. Values are displayed as fold change over input, normalized to the GAPDH negative control. Data are included from 3 biological replicates. (B-D) TREX-BCBL-1 cells were nucleofected with control scramble (scr) siRNAs or siRNAs specific to METTL3, YTHDF2, or YTHDF3, then lytically reactivated by treatment with dox, TPA and ionomycin for 72 hr. (B) Knockdown efficiency of the m⁶A proteins relative to the loading control GAPDH was visualized by western blot. (C) Levels of the KSHV ORF50 and ORF59 proteins were assayed by western blot in the control and m⁶A protein-depleted samples. Additional replicates are shown in S4 Fig. (D) ORF50 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs and reactivated for 36 hr with dox, TPA and ionomycin. (E) Viral supernatant from the reactivated control or m⁶A protein depleted TREX-BCBL-1 cells was transferred to uninfected HEK293T recipient cells, whereupon transfer of infection was quantified by RT-qPCR for the viral LANA transcript 48 hr post supernatant transfer. Individual data points represent 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values <0.05, with *** representing P value ≤ 0.001.