HSV ICP27 hijacks host splicing factor SRSF3 to regulate pre-mRNA splicing and export for viral gene expression and...

HSV ICP27, a multifunctional essential immediate early (IE) viral protein, regulates both viral and host pre-mRNA processing in a gene/sequence-specific manner. Using viral mutagenesis studies, we investigated the mechanisms underlying ICP27-mediated co-transcriptional splicing inhibition. We report that ICP27 inhibits pre-mRNA splicing by hijacking host serine/arginine-rich splicing factor 3 (SRSF3), which binds to an exonic ICP27/SRSF3-responsive motif near the 5' splice site of targeted transcripts, revealing that interaction with SRSF3 enhances both target specificity and efficiency of ICP27-mediated aberrant splicing, in a way independent of its RNA-binding RGG domain. Furthermore, ICP27 co-opts SRSF3 to promote nuclear-export of unspliced mRNA targets via the nuclear RNA export factor 1 (NXF1). Viruses with mutations both in the ICP27 N-terminal nuclear export signal (NES), via which ICP27 interacts with NXF1, and in the RGG RNA-binding domain, are defective in ICP27-mediated splicing inhibition, expression of ICP27-dependent genes, and viral growth, revealing that ICP27-mediated nuclear export of unspliced mRNA is indispensable for ICP27-mediated splicing inhibition and gene expression. Preventing U1 small nuclear ribonucleoprotein (U1 snRNP) binding by knockdown of U1-70K, a component of U1 snRNP that binds to the 5' splice site, led both to splicing inhibition and to enhanced expression of ICP27-dependent genes. Together, these results suggest a spatiotemporal role for ICP27 in regulating sequence-specific pre-mRNA splicing by hijacking SRSF3, preventing spliceosome formation, and subsequently promoting nuclear export of aberrantly processed mRNAs containing restrictive elements including intact 5' splice sites, which would otherwise be detained, spliced or degraded in the nucleus. We hypothesize that during latency, HSV likely takes advantage of the host mRNA processing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion when ICP27 is absent during latency. Upon reactivation, ICP27 is essential for ensuring both the quality and quantity of viral gene expression, enabling optimal viral replication.