Supplementary file 1_Generation of iVero.219-mcRTA: a doxycycline-inducible high-titer KSHV producer cell line with multicopy orf50 integration.doc

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus whose reactivation from latency to lytic replication is orchestrated by the replication and transcription activator (RTA) encoded by orf50. Doxycycline (DOX)-inducible RTA expression is commonly used in engineered cellular models to trigger this reactivation and produce infectious virus. However, this approach often requires co-treatment with histone deacetylase inhibitors (HDACi), such as sodium butyrate (SB), whose pleiotropic epigenetic effects can obscure experimental interpretation. To address this limitation, we generated the iVero.219-mcRTA polyclonal cell line by sequential lentiviral transduction using vectors with distinct antibiotic selection markers, achieving stable multicopy integration of the inducible RTA cassette, with total copy number reaching approximately 150 per cell as determined by droplet digital PCR. Upon DOX induction alone, 51.6% of cells expressed RFP, indicating efficient reactivation. qPCR quantification of viral DNA in the supernatant showed (5.7 ± 0.1) × 104 genome copies/mL, and infection assays in 293T cells yielded an infectious titer of (2.4 ± 0.1) × 104 IU/mL based on Poisson distribution analysis of serial dilutions. Supplementation with SB enhanced these values to (1.5 ± 0.1) × 105 genome copies/mL and (7.5 ± 0.2) × 104 IU/mL, respectively. Notably, the supernatant induced with DOX alone, once concentrated, achieved an infectious titer of (2.1 ± 0.1) × 105 IU/mL, without the need for HDACi. Collectively, the iVero.219-mcRTA system enables robust, high-titer KSHV production using DOX alone, providing a simplified and HDACi-independent tool that will facilitate future KSHV research.