KSHV Reprograms Host RNA Splicing via FAM50A to Activate STAT3 and Drive Oncogenic Cellular Transformation

RNA alternative splicing is a fundamental cellular process implicated in cancer development. Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent of multiple human malignancies, including Kaposi’s sarcoma (KS), remains a significant concern, particularly in AIDS patients. A CRISPR-Cas9 screening of matched primary rat mesenchymal stem cells (MM) and KSHV-transformed MM cells (KMM) identified key splicing factors involved in KSHV-induced cellular transformation. To elucidate the mechanisms by which KSHV-driven splicing reprogramming mediates cellular transformation, we performed transcriptomic sequencing, identifying 131 differential alternative splicing transcripts, with exon skipping as the predominant event. Notably, these transcripts were enriched in vascular permeability, multiple metabolic pathways and ERK1/2 signaling cascades, which play key roles in KSHV-induced oncogenesis. Further analyses of cells infected with KSHV mutants lacking latent genes including vFLIP, vCyclin and viral miRNAs, as well as cells overexpressing LANA, revealed their involvement in alternative splicing regulation. Among the identified splicing factors, FAM50A, a component of the spliceosome complex C, was found to be crucial for KSHV-mediated transformation. FAM50A knockout resulted in distinct splicing profiles in both MM and KMM cells, and significantly inhibited KSHV-driven proliferation, cellular transformation and tumorigenesis. Mechanistically, FAM50A knockout altered SHP2 splicing, promoting an isoform with enhanced enzymatic activity that led to reduced STAT3 Y705 phosphorylation in KMM cells. These findings reveal a novel paradigm in which KSHV hijacks host splicing machinery, specifically FAM50A-mediated SHP2 splicing, to sustain STAT3 activation and drive oncogenic transformation. RNA-seq profiling of MM cells and KMM cells and their FAM50A knockout cell lines (MM-sgFAM50A-11, MM-sgFAM50A-12, MM-sgFAM50A-14, KMM-sgFAM50A-15, KMM-sgFAM50A-22, KMM-sgFAM50A-28ko).