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. 2017 Jul 27;91(16):e00466-17.
doi: 10.1128/JVI.00466-17. Print 2017 Aug 15.

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

Fengchun Ye  1 , E Ricky Chen  2 , Timothy W Nilsen  3
Affiliations

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N6-Adenosine Methylation To Promote Lytic Replication

"VSports app下载" Fengchun Ye et al. J Virol. .

Abstract

N6-adenosine methylation (m6A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m6A modification. The levels of m6A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m6A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m6A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m6A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10 VSports手机版. Interestingly, RTA induced m6A and enhanced its own pre-mRNA splicing. Our results not only demonstrate an essential role of m6A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m6A machinery to its advantage in promoting lytic replication. IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m6A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication. .

Keywords: KSHV; N6-adenosine methylation; RNA splicing; lytic replication V体育安卓版. .

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Figures

FIG 1
FIG 1
MeRIP–qRT-PCR measurement of m6A-mRNA and total mRNA of KSHV transcripts. (A) Schematic presentation of MeRIP procedure. (B) Melting temperature (Tm) of qRT-PCR product of KSHV ORF50 (RTA). Positive signals were seen only with cDNAs from the input and the product of RIP with anti-m6A (RIP-m6A). No signal was seen with cDNAs from the product of RIP with control IgG (RIP-IgG). (C and D) Percentages of m6A-mRNA of β-actin in the products of RIP with m6A (C) and IgG (D), noting that the levels of m6A-mRNA of β-actin from BCBL1 cells treated with PBS or TPA for 24 h were several hundred times higher in the products of RIP with m6A than in the products of RIP with IgG.
FIG 2
FIG 2
The levels of total mRNA and m6A-mRNA of KSHV lytic transcripts increase in parallel when cells are stimulated for lytic replication. (A) Levels of total mRNA and m6A-mRNA of the 5.8-kb and 5.40-kb tricistronic latent transcripts encoding LANA (ORF73), viral cyclin (ORF72), and viral FLIP (ORF71) and the 1.7-kb bicistronic latent transcript encoding viral cyclin (ORF72) and viral FLIP (ORF71) in BCBL1 cells treated with PBS (placebo control) or TPA (20 ng/ml) for 24 h. (B) Levels of total mRNA and m6A-mRNA of lytic transcripts ORF45, ORF49, ORF50 (RTA), ORF57, ORF59, and ORFK8 from the cells described in the legend to panel A. All qRT-PCRs were conducted in triplicate. The statistical significance of the differences in the level of m6A-mRNA or total mRNA of a given transcript between cells treated with PBS and cells treated with TPA was analyzed by an unpaired t test. *, differences with P values of <0.05 (n = 3).
FIG 3
FIG 3
Levels of total mRNA and m6A-mRNA of KSHV IE transcripts ORF45 and ORF50 (RTA) and late transcripts ORF63 and ORF75 in BCBL1 cells at different times after TPA treatment. hpt, hours posttreatment.
FIG 4
FIG 4
Posttranscriptional m6A modification of KSHV transcripts also occurs in endothelial cells and can be induced by different lytic replication stimuli. (A) Levels of total mRNA and m6A-mRNA of KSHV lytic transcripts ORF50 (RTA) and ORF57 in TIVE-KSHV cells treated with PBS or TPA for 24 h. (B) Levels of total mRNA and m6A-mRNA of ORF50 (RTA) in BCBL1 cells treated with PBS (placebo), TPA (20 ng/ml), H2O2 (400 μM), NaB (0.5 mM), or TNF-α (10 ng/ml) for 24 h. The statistical significance of the differences in the level of m6A-mRNA or total mRNA of a given transcript between cells treated with PBS and cells treated with different stimuli was analyzed by an unpaired t test. *, differences with P values of <0.05 (n = 3).
FIG 5
FIG 5
shRNA KD of FTO increases m6A and KSHV lytic gene expression. (A) Levels of FTO mRNA in BCBL1 cells expressing FTO-specific shRNA from Santa Cruz Biotechnologies (shRNA-SC) or Origene Technologies, Inc. (shRNA-OT), or control shRNA. The cells were treated with PBS or TPA for 24 h. (B and C) Levels of ORF50 (RTA) (B) and ORF57 (C) mRNAs in the cells described in the legend to panel A. (D) Levels of m6A-mRNA of ORF50 (RTA) in the cells described in the legend to panel A. (E) Western blot detection of FTO and KSHV lytic proteins encoded by ORF50 (RTA) and ORF57 in the cells described in the legend to panel A. The level of the housekeeping gene β-tubulin was used as a loading control. (F) Relative levels of KSHV virions in the supernatants of the cells described in the legend to panel A at 96 h after TPA stimulation, determined by quantitative PCR using primers specific for ORF72. The cellular debris in the supernatants was removed by high-speed centrifugation (4,000 × g, 15 min), followed by filtration through 0.8-μm-pore-size filters. Total DNAs from 200 μl of each supernatant and 200 μl of the corresponding cells were purified by using a Qiagen genomic DNA purification kit. The level of viral DNA in each supernatant was normalized to that of the corresponding cellular DNA measured with primers specific for β-actin. The level of viral DNA in the supernatant from cells expressing control shRNA and treated with PBS was set as a reference and was equal to 1, and the relative level (fold change) of viral DNA in any of the other supernatants was calculated by using the formula 1/2ΔCT, where ΔCT is the difference in the CT values after normalization between the supernatant in question and that of the reference. All quantitative PCRs were carried out in triplicate.
FIG 6
FIG 6
KD of METTL3 decreases m6A and reduces KSHV lytic gene expression. (A) Levels of METTL3 mRNA in BCBL1 cells expressing METTL3-specific shRNA from Santa Cruz Biotechnologies (shRNA-SC) or Origene Technologies, Inc. (shRNA-OT), or control shRNA. The cells were treated with PBS or TPA for 24 h. (B and C) Levels of ORF50 (RTA) (B) and ORF57 (C) mRNA in the cells described in the legend to panel A. (D) Levels of m6A-mRNA of ORF50 (RTA) in the cells described in the legend to panel A. (E) Western blot detection of METTL3 and KSHV lytic proteins encoded by ORF50 (RTA) and ORF57 in the cells described in the legend to panel A. The level of β-tubulin was used as a loading control. (F) Relative levels of KSHV DNA in the supernatants of the cells described in the legend to panel A at 96 h after TPA stimulation, which were determined as described in the legend to Fig. 5F.
FIG 7
FIG 7
Inhibition of FTO activity enhances KSHV lytic gene expression, while blocking of m6A abolishes lytic gene expression and virion production. (A and B) Relative levels of KSHV latent transcripts (5.4 kb and 5.8 kb) and lytic transcripts ORF50 (RTA) and ORF57 in BCBL1 cells treated with PBS (control), MA (1 μM), DAA (25 μ), and TPA, alone or in combination, for 24 h. (C) Levels of m6A-mRNA of ORF50 (RTA) and ORF57 in the BCBL1 cells described in the legend to panels A and B. (D) Western blot detection of KSHV latent protein LANA (ORF73) and lytic proteins encoded by ORF50 (RTA), ORF57, ORF62, and ORF65 in cells treated as described in the legend to panels A and B for 24 h and 72 h. (E) Representative images of HUVECs at 72 h postinfection with culture supernatants from equal numbers of BCBL1-BAC36 cells that were stimulated as described in the legend to panels A and B and collected at 5 days after treatment. (F) Percentage of GFP-positive cells at 72 h postinfection with the different culture supernatants described in the legend to panel E.
FIG 8
FIG 8
Blocking of m6A inhibits ORF50 (RTA) pre-mRNA splicing. (A) Schematic presentation of the tricistronic pre-mRNA encoding ORF50 (RTA), ORFK8, and ORFK8.1, as well as the locations (indicated with arrows) of the primers used for ORF50 (RTA)-specific cDNA synthesis and qRT-PCR detection of RTA pre-mRNA and mRNA. (B) Levels of ORF50 (RTA) pre-mRNA and mRNA in BCBL1 cells treated with PBS (placebo), TPA, or TPA plus DAA for 24 h. (C) ORF50 (RTA) mRNA-to-pre-mRNA ratios in the cells described in the legend to panel B.
FIG 9
FIG 9
Specific m6A sites in ORF50 (RTA) pre-mRNA are responsible for splicing. (A) m6A sites in the ORF50 (RTA) locus determined by MeRIP-seq. (B) Genomic locations of m6A consensus GGAC sites in the ORF50 (RTA) locus of the KSHV genome and presentation of the pExon1-intron-exon2-GFP plasmid. (C) ORF50 (RTA) mRNA-to-pre-mRNA ratios in 293T cells transfected with equal amounts (4 μg) of wild-type (WT) pExon1-intron-exon2-GFP or its mutants with mutations at each of the individual m6A sites (Mut-A to Mut-J) (GGAC → GGCC). (D) Western blot detection at 48 h posttransfection of RTA and β-tubulin in the 293T cells described in the legend to panel C. (E) Representative images of GFP expression in the cells described in the legend to panel C. The statistical significance of the differences in the ORF50 (RTA) mRNA-to-pre-mRNA ratio between cells transfected with the wild-type plasmid and cells transfected with a plasmid harboring any mutant was analyzed by an unpaired t test. *, differences with P values of <0.05 (n = 3).
FIG 10
FIG 10
The m6A nuclear reader YTHDC1 is associated with RNA splicing factors SRSF3 and SRSF10 in BCBL1 cells. Equal amounts (1 mg) of proteins prepared from BCBL1 cells treated with PBS, TPA, or TPA plus DAA for 24 h were used for coimmunoprecipitation (co-IP) with a rabbit anti-YTHDC1 antibody or control IgG. The co-IP products and input samples were subsequently analyzed by Western blot detection with antibodies to YTHDC1, SRSF3, and SRSF10.
FIG 11
FIG 11
RIP-qRT-PCR measurement of RNA bound by YTHDC1, SRSF3, and SRSF10. (A) Schematic presentation of RIP–qRT-PCR procedure. (B) Locations of the primers used for qRT-PCR measurement of a protein-bound RNA fragment carrying m6A site A in ORF50 (RTA) pre-mRNA, as well as th eprimers used for detection of ORF50 (RTA) mRNA. (C) RIP products obtained by qRT-PCR analyzed in an agarose (2%) gel by electrophoresis. The ∼100-bp fragment (RTA mRNA) was detected only in the input (before RNase A digestion), suggesting that it was not protected by RNA binding proteins and, thus, was sensitive to RNase A digestion. In contrast, the ∼150-bp fragment (RTA pre-mRNA in the m6A site A region) was protected and could be pulled down by anti-YTHDC1 antibody but not control IgG.
FIG 12
FIG 12
m6A modification of site A in ORF50 (RTA) pre-mRNA is required for recruitment of YTHDC1, SRSF3, and SRSF10. (A) Location of the m6A site A region analyzed by RIP–qRT-PCR with antibodies to YTHDC1, SRSF3, and SRSF10 and the specific primers listed in Table 2. (B) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and the levels of m6A in the site A region in BCBL1 cells treated with PBS or TPA for 24 h. For all comparisons, the levels of RNA and m6A in cells treated with PBS were set equal to 1. (C) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and the levels of m6A in the site A region in 293T cells transfected with wild-type (WT) pExon1-intron-exon2-GFP or its mutant with site A mutated (Mut-A). For all comparisons, the levels of RNA and m6A in cells transfected with the wild-type plasmid were set equal to 1. *, differences with a P value of <0.05.
FIG 13
FIG 13
m6A modification of site F in ORF50 (RTA) pre-mRNA is required for recruitment of YTHDC1 and SRSF3. (A) Location of m6A site F region analyzed by RIP–qRT-PCR with antibodies to YTHDC1, SRSF3, and SRSF10 and the specific primers listed in Table 2. (B) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and the levels of m6A in the site F region in BCBL1 cells treated with PBS or TPA for 24 h. For all comparisons, the levels of RNA and m6A in cells treated with PBS were set equal to 1. (C) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and the levels of m6A in the site F region in 293T cells transfected with wild-type (WT) pExon1-intron-exon2-GFP or its mutant with site F mutated (Mut-F). For all comparisons, the levels of RNA and m6A in cells transfected with the wild-type plasmid were set equal to 1. *, differences with a P value of <0.05.
FIG 14
FIG 14
m6A modification of site G in ORF50 (RTA) pre-mRNA is required for recruitment of YTHDC1 and SRSF3 and disassociation of SRSF10. (A) Location of the m6A site G region analyzed by RIP–qRT-PCR with antibodies to YTHDC1, SRSF3, and SRSF10 and the specific primers listed in Table 2. (B) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and the levels of m6A in the site G region in BCBL1 cells treated with PBS or TPA for 24 h. For all comparisons, the levels of RNA and m6A in cells treated with PBS were set equal to 1. (C) Relative levels of RNA bound by YTHDC1, SRSF3, or SRSF10 and levels of m6A in the site G region in 293T cells transfected with wild type (WT) pExon1-intron-exon2-GFP or its mutant with site G mutated (Mut-G). For all comparisons, the levels of RNA and m6A in cells transfected with the wild-type plasmid were set equal to 1. *, differences with a P value of <0.05.
FIG 15
FIG 15
KSHV lytic switch protein RTA (ORF50) induces m6A and enhances its own pre-mRNA splicing. (A) Western blot detection of RTA and β-tubulin in iSLK-BAC16 cells without (Control) and with doxycycline stimulation for 24 h and 293T cells at 48 h posttransfection with an equal amount (4 μg) of pRTA-3×FLAG (pRTA) or the empty vector. (B) Dot blot detection of m6A in 10 μg total RNAs isolated from the cells described in the legend to panel A, using an antibody to m6A and subsequent chemiluminescence detection. The blot was also stained with ethidium bromide (EtBr). (C) Relative levels of total mRNA and m6A-mRNA of ORF50 (RTA) in iSLK-BAC16 cells treated as described in the legend to panel A. (D) ORF50 (RTA) mRNA-to-pre-mRNA ratios in 293T cells cotransfected with wild-type pExon1-intron-exon2-GFP (WT; 2 μg) plus pRTA-3×FLAG (pRTA; 2 μg) or the empty vector (Vector; 2 μg) or mutant plasmids with site A or F mutated (Mut-A and Mut-F, respectively) with similar cotransfection combinations. *, differences that were statistically significant with P values of <0.05.
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