Bioinformatic Analysis of KSHV miRNAs binding to human AID.

<p>Columns identify miRNA name, position within AID mRNA where predicted binding of seed sequence occurs, type of target site and miRNA:gene interaction score according to Bartel D.P. (2009). miRNAs are ordered according the score, from strongest to weakest predicted interaction.</p

KSHV-encoded miRNAs directly target 3′UTR of AID.

<p>(<b>A</b>) HEK-293 cells were co-transfected with AID 3′UTR dual luciferase reporter and the indicated miRNA mimic or scrambled negative control miRNA. Renilla luciferase activity was normalized to firefly luciferase activity and then normalized to the average activity of the empty control reporter. Error bars (SD) are derived from triplicates. Scrambled control is set to 1, while miR-155 serves as a positive control. Shown is one representative experiment out of three performed. (<b>B</b>) A schematic representation of the full length <i>AID</i> mRNA and the predicted binding sites for miR-K12-5 and miR-K12-11; specific 3′UTR locations noted in prentices. (<b>C</b>) Relative AID 3′UTR luciferase reporter activity comparing transfection of a single miRNA, miR-K12-5 or miR-K12-11 versus both miRNAs, each at half of the original concentration. (<b>D</b>) Comparison of relative luciferase activity between WT and mutant 3′UTR AID reporters. Double mut combines mutations of mut 3′UTR (950) and (1051). Each bar represents fold change in reporter activity relative to scrambled control. Left panel represents co-transfection of reporter with miR-K12-5, right panel – with miR-K12-11. Statistically significant differences are indicated (*p<.01 by two-tailed, paired student t-test).</p

KSHV infection results in upregulation of AID in Primary Human Tonsillar B cells.

<p>(<b>A</b>) Primary tonsillar cells were infected with KSHV by co-culture with reactivated iSLK.219 for the indicated time. Percent infection of B cells was assessed via flow cytometry by gating on CD19⁺ cells and quantifying infected, GFP⁺ cells (right rectangular gate). (<b>B</b>) Intracellular AID expression was assessed in infected, GFP⁺ B cells (red) and uninfected, GFP⁻ B cells (filled gray) using flow cytometry. Black histogram represents unstained control. (<b>C</b>) Tonsillar cells were infected by co-culture with reactivated iSLK.219 cells for 2 days. CD19⁺ population was enriched (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003748#ppat.1003748.s001" target="_blank">figure S1</a>) and subsequently sorted for infected, GFP⁺ and uninfected, GFP⁻ cells. The level of <i>AID</i> transcript was measured by qRT-PCR. Shown are mean relative values ± SD from 4 independent patients (*p<.01 by two-tailed, paired student t-test). (<b>D</b>) Tonsillar cells were transiently transfected with vFLIP (red) or vector control (gray) plasmids. 48 hrs post transfection CD19⁺, GFP⁺ (transfection marker) cells were analyzed for intracellular AID by flow cytometry. Black histogram represents unstained control.</p

AID expression does not affect viral latency, but results in lytic reactivation and infectivity defects.

<p>(<b>A</b>) BCBL-1 cell were transduced either with empty vector ctr (filled gray) or AID (red) retroviral constructs, selected for 1 wk until all untransduced cells have died. AID protein expression assessed by flow cytometry. Unstained control is shown in black. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003748#ppat.1003748.s002" target="_blank">figure S2A</a> & B. (<b>B, C</b>) Empty vector control or AID-expressing BCBL-1 cells were analyzed for <i>LANA</i> transcript by qRT-PCR (B) or protein by immunofluorescence staining (C) at 10 wks post selection. Error bars (SD) are derived from triplicates. Shown is one representative experiment out of three performed. LANA staining is depicted in green, DAPI nuclear marker is depicted in blue. (<b>D, E</b>) BCBL-1 cells stably transduced with empty vector control or AID for 1 wk (<b>D</b>); 10 wks (<b>E</b>) were reactivated using NaBut. Expression of lytic transcript <i>RTA</i> was analyzed by qRT-PCR at 24 or 48 hrs post reactivation. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003748#ppat.1003748.s002" target="_blank">figure S2C</a>&D; Error bars (SD) are derived from triplicates. (<b>F</b>) Supernatant from cells in (E) was concentrated and 50 µl were used to quantify total KSHV genome copies (using LANA specific primers) by qPCR. (<b>G</b>) Serial dilutions of viral supernatant from (F) were used to infect 3×10⁵ WT HFF cells. Cells were trypsinized and passaged once to remove any uninternalized virus prior to harvesting total gDNA. qPCR was used to quantify average KSHV genome copies per HFF cell (LANA/AID gDNA). Plotted are triplicate averages fitted by linear regression. (<b>H</b>) shRNA was used to knock-down expression of UNG2 in empty vector control or AID-expressing BCBL-1 cells. 1 wk post selection cells were reactivated with NaBut for 24 and 48 hrs and analyzed for induction of <i>RTA</i> by qRT-PCR. (<b>I</b>) BCBL-1 cells were first stably transduced with vector overexpressing UNG2 or empty control, then with AID or empty vector control as in (A). At 6 wks post selection the four stable cell lines were reactivated with NaBut and assessed for infectivity same as in (G).</p

KSHV miRNAs downregulate AID in the context of the entire viral genome.

<p>(<b>A</b>) HEK-293 cells were stably transduced with constructs encoding full length <i>AID</i> mRNA (hAID mRNA) or open reading frame of <i>AID</i> (hAID ORF) and transiently transfected with either WT KSHV BAC (red) or BAC lacking miRNA cluster, ΔmiR BAC(filled gray). Intracellular AID expression was analyzed by flow cytometry at 48 hrs post transfection. Dashed black histogram reflects background staining of AID-negative WT HEK-293 cells. (<b>B</b>) Quantification of AID downregulation upon WT KSHV BAC transfection relative to ΔmiR BAC at 24, 48 or 72 hrs post transfection. Data represent at least three independent experiments for each time point. Dashed bars represent cells stably expressing hAID ORF, solid gray bars represent cells stably expressing hAID mRNA. The percentage of AID downregulation (% MFI), in cells transfected with WT KSHV BAC was calculated relative to cells transfected with the ΔmiR BAC after subtracting background MFI. Shown are mean values ± SD (*p<.05 by two-tailed, paired student t-test).</p

KSHV infection leads to upregulation of NKG2D ligands.

<p>(<b>A</b>) Tonsillar cells were infected by co-culture with reactivated iSLK.219 cells. CD19⁺ population was enriched and subsequently sorted for infected, GFP⁺ and uninfected, GFP⁻ cells. Relative levels of four detected NKG2D transcripts were measured by qRT-PCR. Shown are mean values ± SD from at least three patients (*p<.01 by two-tailed, paired student t-test). (<b>B</b>) CD19⁺ tonsillar B cells were assessed for surface expression of MICB (top panel) and ULBP2 (bottom panel) by flow cytometry. Infected, GFP⁺ cells are depicted in red and uninfected, GFP⁻ cells in filled gray; isotype control is shown in dashed black. (<b>C</b>) Infected cells were treated with DNA damage inhibitors, caffeine (middle panel) and SB218078 (right panel) for the duration of the infection. Infected, drug-treated B cells are shown in blue and uninfected drug-treated cells in thin black as compared to vehicle controls depicted same as in (B). (<b>D</b>) Tonsillar B cells were sorted for infected, GFP⁺ and uninfected, GFP⁻ cells. GFP⁻, uninfected cells were labeled with CFSE for tracking. Infected or uninfected cells were co-incubated with effector NK-92 cells at specified ratios for 5 hours and assayed for killing by flow cytometry. Percentage of dead target cells is indicated by the blue rectangular gates. All experiments presented in the figure were done on day 4 post infection.</p

miR-K12-5 and miR-K12-11 target full length AID for downregulation when expressed at physiological levels.

<p>(<b>A</b>) HEK-293 cells were stably transduced with shRNA constructs expressing individual indicated KSHV miRNAs or negative control miRNA. Total RNA was harvested from the stable cell lines or BC-1 and BCBL-1 cell lines latently infected with KSHV for Northern blot analysis of miRNA expression. Probe binding to miR-K12-5, miR-K12-11 or siRNA against luciferase gene is indicated above each panel. U6 expression serves as a loading control. (<b>B</b>) HEK-293 cells were stably transduced with full length <i>AID</i> mRNA along with the indicated shRNA construct. Total protein was harvested and AID expression detected by Western blot. GAPDH serves as a loading control. (<b>C</b>) Total RNA was isolated from the same cells as described in (B). Relative <i>AID</i> expression was quantified by qRT-PCR analysis. Error bars (SD) are derived from triplicates.</p

Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents

Cyclin G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo­[5,4-<i>b</i>]­pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Cocrystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV life cycle (i.e., viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer, and Parkinson’s disease)