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

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. 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.