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. 2009 Mar 13;5(3):e1000332. doi: 10.1371/journal.ppat.1000332

Figure 7. Knockdown of KIF3A and KHC by short-hairpin RNA (shRNA)–mediated silencing and the effects on KSHV egress and extracellular viral titers.

Figure 7

(A) Inhibition efficiency of KIF3A and KHC expression in BCBL-1 cells. Two sets of shRNA constructs, each consisting of four individual shRNA lentiviral vectors in pLKO.1-puro plasmids against different target sites of KIF3A (KIF3A shRNA clone #s 1–4) and KHC (KHC shRNA clone #s 1–4), respectively, and a non-targeting control shRNA that activates the RNAi pathway without targeting any known human gene were introduced into BCBL-1 cells through lentiviral transduction in the presence of polybrene. Cells were selected through puromycin (2 µg/ml) selection for a week and then tested for effective knockdown of the respective proteins by a Western blot (WB) performed on the whole cell extracts using anti-KIF3A (rabbit-polyclonal, left panel) and KHC (rabbit-polyclonal, right panel). A WB for β-actin protein detection was also performed as an internal loading control. (B) Effects of KIF3A and KHC silencing on KSHV egress. BCBL-1 cells stably expressing the control shRNA, KIF3A shRNAs (clones #s 1–4), and KHC shRNAs (clone #s 1–3) were treated with TPA (20 ng/ml) to induce KSHV lytic replication. Four days post-induction, extracellular KSHV virions were harvested and concentrated 100-fold. Virus stocks were treated with Turbo DNase I, and viral genomic DNA were extracted. KSHV genomic DNA concentration was estimated by a real-time PCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene. Viral titers were presented as viral DNA copy numbers per milliliter (ml) of the supernatant. (C) Effects of lentivirus transduction on KSHV viral DNA replication. BCBL-1 cells stably expressing the KIF3A and KHC shRNAs were treated with TPA (20 ng/ml) to induce KSHV lytic replication. Two days post-induction, cells were collected and total intracellular DNAs were extracted with the Qiagen DNeasy kit. Intracellular viral DNAs were measured by a real-time PCR with primers directed to the ORF73 gene from a 100 ng input DNA. The viral genome copies were normalized to 20,000 copies of GAPDH.