We wished to examine the antiviral activity of nelfinavir in greater detail using a more tractable cell culture system for KSHV. To this end we re-created an inducible SLK cell line harboring KSHV, similar to iSLK.219 cl.10 that was originally described by Myoung and Ganem [
28]. The KSHV recombinant rKSHV.219 was originally isolated by Jeff Vieira [
26]. This virus expresses GFP constitutively (EF1a promoter) and upon lytic induction expresses RFP driven by the lytic KSHV PAN promoter. It also carries a puromycin resistance gene. Previously, we had obtained the Vero rKSHV-219 cell line from Jeff Vieira. This line requires infection with a recombinant baculovirus expressing ORF50 (RTA) and sodium butyrate treatment for lytic induction [
26]. This was not conducive for our experiments. Therefore, we derived rKSHV.219 virus from the Vero cell line (after lytic induction) and infected iSLK cells. These cells express RTA under control of a doxycycline (DOX)-responsive promoter, the tetracycline-responsive element (TRE), for more efficient lytic induction [
28]. Following infection of iSLK cells we derived cell lines that were GFP positive and puromycin resistant. Individual clones were selected for their lytic inducibility (RFP fluorescence) following the addition of doxycycline. One such clonal cell line designated 5r219 was used for all our experiments. Upon the addition of doxycycline (DOX) the cells began to express RFP indicating induction of lytic gene expression (Fig.
1A). We quantitated virus production from these cells following lytic induction (+ doxycycline) using a GFP plating assay on Vero and HEK-293T monolayers (Fig.
1B). The GFP fluorescence assay is a measure of biological infectivity in that GFP is only expressed after KSHV virus enters and begins to replicate in these cells. This assay demonstrated significant virus production from the 5r219 cells and because we observed similar results using either Vero or HEK-293T cells, we used Vero cells for subsequent plating assays. We chose to use a qPCR assay to quantitate viral genomes produced by 5r219 cells. The first experiments, shown here, were used to demonstrate the production of KSHV virus following induction with DOX (1 µg/mL) alone or with DOX and 1 mM sodium butyrate (DOX/NB) (Fig.
1C). This was also visually observed when we infected Vero cell monolayers with virus harvested from 5r219 cultures at 72 h post-induction. The GFP signal indicative of KSHV infectious virus was high for both induced cultures (DOX or DOX/NB) (Fig.
1D). Because these observations showed that we could achieve significant virus yields at 3 days post-induction with either induction method, we thus eliminated sodium butyrate from the induction because of the toxicity of this compound. We also compared the qPCR and GFP titration methods using virus harvested (72 h) from 5r219 (DOX or DOX + NB) induced cells (Fig.
S1). While the GFP assay is a visual reporter of infectious virus, it was not as easily quantifiable compared to the qPCR assay because of the non-uniformity of the appearance of the GFP fluorescence.