RNA N6-adenosine methylation (m6A) steers epitranscriptomic control of herpesvirus replication

Latency is a hallmark of all herpesviruses, during which the viral genomes are silenced through DNA methylation and suppressive histone modifications. When latent herpesviruses reactivate to undergo productive lytic replication, the suppressive epigenetic marks are replaced with active ones to allow for transcription of viral genes. Interestingly, by using Kaposi’s sarcoma-associated herpesvirus (KSHV) as a model, we recently demonstrated that the newly transcribed viral RNAs are also subjected to post-transcriptional N6-adenosine methylation (m6A). Blockade of this post-transcriptional event abolishes viral protein expression and halts virion production. We found that m6A modification controls RNA splicing, stability, and protein translation to regulate viral lytic gene expression and replication. Thus, our finding for the first time reveals a critical role of this epitranscriptomic mechanism in the control of herpesviral replication, which shall shed lights on development of novel strategies for the control of herpesviral infection.

latency through DNA methylation, repressive histone modifications, and other negative gene expression regulatory mechanisms [7][8][9][10][11] . When the latent virus reactivates, prompt epigenetic changes occur, leading to transactivation of the viral genome. However, our recent study discovered that KSHV reactivation stalls if the newly transcribed viral RNAs fail to undergo post-transcriptional N 6 -adenosine methylation (m 6 A) [12] . Our finding highlights a pivotal role of this epitranscriptomic mechanism in the control of KSHV lytic replication.
Similar to HIV-1, most KSHV transcripts undergo m 6 A modification, and the level of m 6 Amodified mRNA of a given viral transcript increases in parallel with that of total mRNA when latently infected cells are induced by phorbol ester (TPA) or other lytic replication stimuli. Expressional knocking down of the m 6 A writer METTL3 substantially reduces TPA induction of KSHV lytic genes, and blockade of m 6 A reaction literally abolishes expression of all lytic genes examined and halts virion production. In contrast, expressional knocking down or activity inhibition of the m 6 A eraser FTO has the opposite effects.
To understand how RNA methylation controls KSHV replication, we examined the effect of m 6 A on expression of viral regulator of transcription activation (RTA), which, encoded by open reading frame 50 (ORF50), is a key mediator of the switch from latency to lytic gene expression [40] . Due to differential splicing, the ORF50 (RTA) and ORFK8 loci produce at least three different groups of transcripts, including ORF50 /ORFK8/ORFK8.1 tricistronic mRNAs, ORFK8/ORFK8.1 bicistronic mRNAs, and monocistronic ORFK8.1 mRNAs [41] . RTA, which is expressed from the tricistronic mRNAs, consists of two exons and one intron (Fig. 1). Interestingly, blockade of m 6 A substantially reduces the level of TPA-induced RTA mRNA but has much less an effect on the level of RTA pre-mRNA, suggesting that m 6 A controls RTA pre-mRNA splicing. Indeed, multiple m 6 A sites are identified in RTA pre-mRNA. Data from genetic mutation assays demonstrate that the m 6 A sites in the intron near the two splicing sites are critical for RTA expression, and one m 6 A site in Exon2 near the splicing site also plays an important role in RTA pre-mRNA splicing. Data from RNA immuno-precipitation (RIP) assays confirm that these sites are indeed m 6 A modified. In addition, both SRSF3 and SRSF10 are present at the m 6 A sites in the intron near the two splicing sites, and the levels of m 6 A and these splicing factors increase significantly upon TPA treatment. Mutation of these m 6 A sites abolishes the RNA-protein interactions and RTA protein expression, thus suggesting that m 6 A modification of these sites is critical for recruitment of SRSF3 and SRSF10 and exclusion of the intron. In contrast, the m 6 A site in Exon2 near the splicing site is critical for removal of SRSF10 and Exon2 inclusion splicing. Therefore, our data highlight a pivotal role of m 6 A modification in RTA pre-mRNA splicing. Interestingly, when the m 6 A sites in both the intron and Exon2 are simultaneously mutated, the level of RTA pre-mRNA drops dramatically (un-published results), suggesting that m 6 A modification also contributes to stability of RTA pre-mRNA. In addition, similar to host mRNAs and HIV-1 transcripts, m 6 A modification may also promote RTA mRNA stability and protein translation through association with m 6 A readers YTHDF1, YTHDF2, and YTHDF3.
Finally, we also found that expression of RTA protein increases the levels of m 6 A modification and promotes its own pre-mRNA splicing. RTA is known to enhance its own transcription [42] . Thus, our data for the first time demonstrate that RTA increases its own expression through both transcriptional and post-transcriptional mechanisms. Very interestingly, the KSHV latent protein LANA, which inhibits RTA expression to promote latency [43] , suppresses TPA induction of RNA m 6 A modification and inhibits RTA pre-mRNA splicing (un-published results).
In summary, our results not only demonstrate an essential role of m 6 A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved two opposite mechanisms to manipulate the host m 6 A machinery to its advantage in promoting lytic replication and latency respectively. This epitranscriptomic mechanism may be used by other herpesviruses as well. Our findings shall shed light on development of new strategies for the control of herpesviral infection. Multiple m 6 A sites are found in RTA pre-mRNA, which are methylated by m 6 A writers METTL3, METTL14, and WTAP. The m 6 A sites in the intron near the two splicing sites are critical for YTHDC1 binding and recruitment of splicing factors SRSF3 and SRSF10 while the m 6 A site in Exon2 near the splicing site is important for recruitment of SRSF3 and dissociation of SRSF10. Interactions between the m 6 A-modified RTA pre-mRNA and the different splicing factors ensure exclusion splicing of the intron to generate RTA mRNA. The other m 6 A sites may enhance RTA mRNA export, stability, and translation through interaction with m6A readers YTHDF1, YTHDF2, and YTHDF3. The expressed RTA protein enhances the host's m 6 A machinery to increase the levels of m 6 A to promote its own pre-mRNA splicing and KSHV lytic gene expression. In contrast, KSHV latent protein LANA has the opposite effects on m 6 A and RTA pre-mRNA splicing.