Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

Author summaryLike other herpesviruses, the expression of KSHV genes is in a tightly regulated manner during viral infection. The contribution of RNA splicing in the regulation of KSHV gene expression was extensively investigated in this report. We found that approximately one-third of all viral genes express spliced transcripts of which many undergo RNA alternative splicing. We identified a subset of viral RNA splicing events susceptible to the regulation of viral RNA-binding protein ORF57. This led to the discovery of ORF57-mediated regulation of the bicistronic ORF70-K3 RNA splicing and production of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3. Such regulation allows timely but mutually exclusive expression of these two viral proteins to execute their unique roles in immune evasion and viral genome replication during the KSHV lytic cycle. Thus, these data provide new insights into KSHV biology. Finally, our study represents a blueprint for RNA splicing studies of other pathogens in infected cells. RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.