Fig 9.
The ORF59 PM3 mutant of KSHV is defective in virion production. (A and B) Cells transfected with BAC36 ΔORF59 into either empty lentivirus vector, wt ORF59-expressing cells, or PM3-expressing cells were tested for LANA-, RTA-, and HA-tagged ORF59 or PM3 expression. Uninduced cells were shown to express LANA (IB: LANA) and ORF59 and PM3 (IB: HA) but to express very little RTA (IB: RTA). The results for GAPDH (IB: GAPDH) are presented to show equal loading. (B) Cells were induced with TPA and sodium butyrate to induce reactivation, which was confirmed by the detection of RTA (IB: RTA) All these cell lines showed similar levels of LANA RTA expressions. (C) DNA was extracted from uninduced cells to examine the latently maintained genome copies in these cell lines. LANA (ORF73) primers were used to calculate the KSHV genome copy number using the ΔΔCT method of quantitation. The relative number of copies of episomally maintained genomes was plotted by using a vector control as a reference. The results showed maintenance of almost identical numbers of viral episomes in these cell lines. (D) Cells were induced with 20 ng TPA and 1.5 mM sodium butyrate for 5 days, after which the supernatant was collected and virion DNA was extracted. The relative numbers of virions produced were determined by real-time qPCR, which showed a significant decrease in virion production in ORF59 with mutated Ser376, Ser378, and Ser379 (PM3) compared to that in cells with wt ORF59.