Kaposi's sarcoma-associated herpesvirus modulates the pseudouridine pathway to enhance lytic replication

Pseudouridylation is a prevalent RNA modification shown to occur in tRNAs, rRNAs, snoRNAs and most recently mRNAs and ncRNAs. Emerging evidence suggests that this dynamic RNA modification is implicated in altering gene expression by regulating RNA stability, modulating translation elongation and modifying amino acid substitution rates. However, the role of pseudouridylation in infection is poorly understood. Here we demonstrate that Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the pseudouriylation pathway to enhance replication. We show the pseudouridine synthases (PUS), PUS1 and PUS7 are essential for efficient KSHV lytic replication, supported by the redistribution of PUS7 to viral replication and transcription complexes. Here we present a comprehensive analysis of KSHV RNA pseudouridylation revealing hundreds of modified RNAs at single-nucleotide resolution. Notably, we further demonstrate that pseudouridylation of the KSHV-encoded polyadenylated nuclear RNA (PAN) plays a significant role in the stability of PAN RNA and in the association of the KSHV ORF57 protein. Our findings reveal a novel and essential role of pseudouridine modification in the KSHV replication cycle. Overall design: Samples are compared between respective Non-bisulfite control and Bisulfite treated samples and further compared between latent and doxycyline reactivated TREx-BCBL1-RTA cells, identifying deletion signatures indicative of pseudouridine modification presence.