Optimizing Recombinant KSHV Production

To establish efficient recombinant virus-producing cells, tetracycline/doxycycline (Dox)-inducible (Tet-On) RTA/ORF50-expressing iSLK cells [47 (link),48 (link),49 (link)] or iVero cells [50 (link)] were used as recombinant KSHV-producing cells. For maintenance, iSLK cells were cultured in a growth medium containing 1 μg/mL of puromycin (InvivoGen, CA, USA) and 0.25 mg/mL of G418 (Nacalai Tesque, Kyoto, Japan). iVero cells were cultured in a growth medium containing 2.5 μg/mL of puromycin. WT KSHV BAC16 (WT-BAC16) and ORF21-mutated KSHV BAC16 (21KD-BAC16 and 21del-BAC16) were transfected into iSLK and iVero cells by the calcium phosphate method [51 ]. The transfected cells were selected under 1000 μg/mL of hygromycin B (Wako, Osaka, Japan) to establish doxycycline-inducible recombinant KSHV-producing cell lines. iSLK (or iVero) cells transfected with WT-BAC16, 21KD-BAC16, and 21del-BAC16 were named iSLK-WT, iSLK-21KD, and iSLK-21del cells, respectively (or iVero-WT, iVero-21KD, and iVero-21del cells, respectively). To induce the lytic replication for recombinant KSHV production, BAC16-harboring iSLK (or iVero) cells were treated with sodium butyrate (NaB) and Dox. NaB binds to histone deacetylases (HDACs) and induces the hyperacetylation of the histones, resulting in transcriptional activation. Dual treatment of both Dox and NaB was used in this study because it induced the RTA/ORF50 expression and the lytic cycle more effectively.