Lentiviral short hairpin RNA (shRNA) to knock-down KSHV latent genes. (A) The lentiviral construct used for shRNA production. cPPT, central polypurine tract; SFFV, spleen focus forming virus promoter; PAC, puromycin N-acetyltransferase gene; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; LTR, long terminal repeat. (B) Graph showing the correlation between MOI measured in 293t IU (293t cell infectious units) per cell, the number of lentiviral inserts per cell determined by TaqMan qPCR for the lentiviral packaging sequence, and the knockdown of LANA expression compared to β-actin control shown by Western blot. (C) Western blot panel for KSHV latent genes (gene name on left side) in BC-3 cells 7 days (sh-vFLIP and sh-vcyclin) or 14 days (sh-LANA) after infection with short hairpin (top label indicate hairpin target). vFLIP and vcyclin expression is simultaneously inhibited when either gene is targeted. (D) Hypothetical representation of the polycistronic nature of the mRNA species of the oncogenic cluster (adapted from Guasparri et al⁴⁰  and Talbot et al⁶⁵  and findings from RNAi knock-down results). (E) TaqMan qRT-PCR of BC-3 cells 96 hours after infection with a short hairpin showing decrease in the relative abundance of the mRNA species responsible for LANA and vcyclin production when infected with an appropriate inhibitor. (Fi) IFA for LANA (images at 600 × magnification) in BC-3, JSC-1, and HBL-6 cells shows that 10 days after infection with sh-LANA, the majority of cells lose characteristic nuclear stippling indicative of LANA expression. (Fii) Higher magnification (800 × top, 200 × bottom) of BC-3 cell expressing LANA despite presence of knock-down. (G) TaqMan qPCR for the lentiviral packaging signal shows that BC-3 cells positive for LANA (sorted for LANA-FITC) contain comparable levels of the sh-LANA construct to unsorted infected BC-3 cells. Error bars represent the standard error of the mean.