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Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification

View Article: PubMed Central - PubMed

ABSTRACT

Broad spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we use parallel genome-wide high-coverage shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad spectrum antiviral with unexplained cytotoxicity1–3. We show that GSK983 blocks cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduces GSK983 cytotoxicity but not antiviral activity, providing an attractive novel approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Together, our results highlight the distinct advantages and limitations of each screening method for identifying drug targets and demonstrate the utility of parallel knockdown and knockout screens for comprehensively probing drug activity.

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Deoxycytidine (dC) reverses the anti-proliferative effect of GSK983 but not antiviral activity. Uridine (a) and deoxycytidine (b) largely reversed GSK983-induced growth inhibition in K562 cells. For (a) and (b), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of uridine or deoxycytidine. Error bars represent ± standard deviation of 4 biological replicates. (c) GSK983 inhibited replication of luciferase-expressing DENV in A549 cells (black). 1 mM uridine reversed antiviral activity (blue), while 1 mM deoxycytidine did not (red). Error bars represent ± standard deviation of 3 biological replicates. (d) Uridine (blue) and deoxycytidine (red) reversed GSK983-induced growth inhibition in A549 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with no exogenous pyrimidines (control), 1 mM uridine, or 1 mM deoxycytidine and the indicated concentration of GSK983. Error bars represent ± standard deviation of 3 biological replicates. (e) Ribonucleoside (uridine) salvage sustains both RNA virus replication and cellular DNA synthesis. (f) Deoxyribonucleoside (deoxycytidine) salvage sustains cellular DNA synthesis but not RNA virus replication. For (e) and (f), Pyr = pyrimidine, rNuc = ribonucleotides, dNuc = deoxyribonucleotides. (g) Deoxycytidine reversed GSK983-induced S phase cell cycle arrest in K562 cells. Following 24 h treatment with 48 nM GSK983, cells were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h and fixed in 70% EtOH. Cells were stained with Azide-fluor 488 and 7-AAD and analyzed by flow cytometry. Flow cytometry plots depict one of three biological replicates.
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Figure 3: Deoxycytidine (dC) reverses the anti-proliferative effect of GSK983 but not antiviral activity. Uridine (a) and deoxycytidine (b) largely reversed GSK983-induced growth inhibition in K562 cells. For (a) and (b), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of uridine or deoxycytidine. Error bars represent ± standard deviation of 4 biological replicates. (c) GSK983 inhibited replication of luciferase-expressing DENV in A549 cells (black). 1 mM uridine reversed antiviral activity (blue), while 1 mM deoxycytidine did not (red). Error bars represent ± standard deviation of 3 biological replicates. (d) Uridine (blue) and deoxycytidine (red) reversed GSK983-induced growth inhibition in A549 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with no exogenous pyrimidines (control), 1 mM uridine, or 1 mM deoxycytidine and the indicated concentration of GSK983. Error bars represent ± standard deviation of 3 biological replicates. (e) Ribonucleoside (uridine) salvage sustains both RNA virus replication and cellular DNA synthesis. (f) Deoxyribonucleoside (deoxycytidine) salvage sustains cellular DNA synthesis but not RNA virus replication. For (e) and (f), Pyr = pyrimidine, rNuc = ribonucleotides, dNuc = deoxyribonucleotides. (g) Deoxycytidine reversed GSK983-induced S phase cell cycle arrest in K562 cells. Following 24 h treatment with 48 nM GSK983, cells were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h and fixed in 70% EtOH. Cells were stained with Azide-fluor 488 and 7-AAD and analyzed by flow cytometry. Flow cytometry plots depict one of three biological replicates.

Mentions: Guided by the appearance of pyrimidine salvage enzymes among the top sensitizing hits from both genomic screens, we examined the ability of pyrimidine salvage metabolites to reverse the anti-proliferative effect of GSK983 on rapidly dividing cells. Uridine, cytidine, or deoxycytidine supplementation reversed GSK983-induced growth inhibition to varying extents in K562 cells (Fig. 3a,b and Supplementary Fig. 6a,b). However, cellular salvage of exogenous ribonucleosides (uridine and cytidine) can sustain RNA virus replication despite DHODH inhibition40,43. We reasoned that deoxycytidine salvage would support DNA but not RNA virus replication given that ribonucleotides cannot be directly biosynthesized from their 2′-deoxy analogues. This raised the intriguing possibility of using a DHODH inhibitor to block RNA virus replication in combination with a deoxycytidine supplement to reverse the anti-proliferative effect on rapidly dividing cells.


Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
Deoxycytidine (dC) reverses the anti-proliferative effect of GSK983 but not antiviral activity. Uridine (a) and deoxycytidine (b) largely reversed GSK983-induced growth inhibition in K562 cells. For (a) and (b), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of uridine or deoxycytidine. Error bars represent ± standard deviation of 4 biological replicates. (c) GSK983 inhibited replication of luciferase-expressing DENV in A549 cells (black). 1 mM uridine reversed antiviral activity (blue), while 1 mM deoxycytidine did not (red). Error bars represent ± standard deviation of 3 biological replicates. (d) Uridine (blue) and deoxycytidine (red) reversed GSK983-induced growth inhibition in A549 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with no exogenous pyrimidines (control), 1 mM uridine, or 1 mM deoxycytidine and the indicated concentration of GSK983. Error bars represent ± standard deviation of 3 biological replicates. (e) Ribonucleoside (uridine) salvage sustains both RNA virus replication and cellular DNA synthesis. (f) Deoxyribonucleoside (deoxycytidine) salvage sustains cellular DNA synthesis but not RNA virus replication. For (e) and (f), Pyr = pyrimidine, rNuc = ribonucleotides, dNuc = deoxyribonucleotides. (g) Deoxycytidine reversed GSK983-induced S phase cell cycle arrest in K562 cells. Following 24 h treatment with 48 nM GSK983, cells were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h and fixed in 70% EtOH. Cells were stained with Azide-fluor 488 and 7-AAD and analyzed by flow cytometry. Flow cytometry plots depict one of three biological replicates.
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Figure 3: Deoxycytidine (dC) reverses the anti-proliferative effect of GSK983 but not antiviral activity. Uridine (a) and deoxycytidine (b) largely reversed GSK983-induced growth inhibition in K562 cells. For (a) and (b), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of uridine or deoxycytidine. Error bars represent ± standard deviation of 4 biological replicates. (c) GSK983 inhibited replication of luciferase-expressing DENV in A549 cells (black). 1 mM uridine reversed antiviral activity (blue), while 1 mM deoxycytidine did not (red). Error bars represent ± standard deviation of 3 biological replicates. (d) Uridine (blue) and deoxycytidine (red) reversed GSK983-induced growth inhibition in A549 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with no exogenous pyrimidines (control), 1 mM uridine, or 1 mM deoxycytidine and the indicated concentration of GSK983. Error bars represent ± standard deviation of 3 biological replicates. (e) Ribonucleoside (uridine) salvage sustains both RNA virus replication and cellular DNA synthesis. (f) Deoxyribonucleoside (deoxycytidine) salvage sustains cellular DNA synthesis but not RNA virus replication. For (e) and (f), Pyr = pyrimidine, rNuc = ribonucleotides, dNuc = deoxyribonucleotides. (g) Deoxycytidine reversed GSK983-induced S phase cell cycle arrest in K562 cells. Following 24 h treatment with 48 nM GSK983, cells were treated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h and fixed in 70% EtOH. Cells were stained with Azide-fluor 488 and 7-AAD and analyzed by flow cytometry. Flow cytometry plots depict one of three biological replicates.
Mentions: Guided by the appearance of pyrimidine salvage enzymes among the top sensitizing hits from both genomic screens, we examined the ability of pyrimidine salvage metabolites to reverse the anti-proliferative effect of GSK983 on rapidly dividing cells. Uridine, cytidine, or deoxycytidine supplementation reversed GSK983-induced growth inhibition to varying extents in K562 cells (Fig. 3a,b and Supplementary Fig. 6a,b). However, cellular salvage of exogenous ribonucleosides (uridine and cytidine) can sustain RNA virus replication despite DHODH inhibition40,43. We reasoned that deoxycytidine salvage would support DNA but not RNA virus replication given that ribonucleotides cannot be directly biosynthesized from their 2′-deoxy analogues. This raised the intriguing possibility of using a DHODH inhibitor to block RNA virus replication in combination with a deoxycytidine supplement to reverse the anti-proliferative effect on rapidly dividing cells.

View Article: PubMed Central - PubMed

ABSTRACT

Broad spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we use parallel genome-wide high-coverage shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad spectrum antiviral with unexplained cytotoxicity1–3. We show that GSK983 blocks cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduces GSK983 cytotoxicity but not antiviral activity, providing an attractive novel approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Together, our results highlight the distinct advantages and limitations of each screening method for identifying drug targets and demonstrate the utility of parallel knockdown and knockout screens for comprehensively probing drug activity.

No MeSH data available.


Related in: MedlinePlus