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Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

Filone CM, Hanna SL, Caino MC, Bambina S, Doms RW, Cherry S - PLoS ONE (2010)

Bottom Line: As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts.Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C.Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

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High-throughput screen for inhibitors of RVFV infection.A. Schematic of screening strategy used in human and insect cells. Cells are seeded in 384 well plates, subsequently 10µM drug was added. The cells were infected with RVFV, then fixed, stained and imaged using automated microscopy. Automated image analysis and statistical thresholds were used to identify inhibitors of RVFV infection. Candidate drugs are then validated and further characterized. B. 6,000 293T cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.06 for 16 hours. Robust Z scores for infection in mammalian were plotted for each replicate in blue. Each of the four plates is denoted by a different symbol. C. 20,000 S2 cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.02 for 48 hours. Robust Z scores for infection in mammalian were plotted for each replicate in red. Each of the four plates is denoted by a different symbol. D. Annotated categories of 59 candidate RVFV inhibitors from LOPAC screen. * denotes over-represented groups p<0.02. E. Venn diagram of the distribution of small molecules: 31 mammalian (blue), 13 insect (red) and 15 pan-inhibitors (purple). * p<10−16. F. Categories of 15 candidates that block infection of both mammalian and Drosophila cells. * denotes over-represented groups p<0.02.
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pone-0015483-g002: High-throughput screen for inhibitors of RVFV infection.A. Schematic of screening strategy used in human and insect cells. Cells are seeded in 384 well plates, subsequently 10µM drug was added. The cells were infected with RVFV, then fixed, stained and imaged using automated microscopy. Automated image analysis and statistical thresholds were used to identify inhibitors of RVFV infection. Candidate drugs are then validated and further characterized. B. 6,000 293T cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.06 for 16 hours. Robust Z scores for infection in mammalian were plotted for each replicate in blue. Each of the four plates is denoted by a different symbol. C. 20,000 S2 cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.02 for 48 hours. Robust Z scores for infection in mammalian were plotted for each replicate in red. Each of the four plates is denoted by a different symbol. D. Annotated categories of 59 candidate RVFV inhibitors from LOPAC screen. * denotes over-represented groups p<0.02. E. Venn diagram of the distribution of small molecules: 31 mammalian (blue), 13 insect (red) and 15 pan-inhibitors (purple). * p<10−16. F. Categories of 15 candidates that block infection of both mammalian and Drosophila cells. * denotes over-represented groups p<0.02.

Mentions: Next, we used this assay platform to screen for small molecule inhibitors of RVFV MP12 infection both in human (293T) and Drosophila (S2) cells (Schematic Figure 2A). We screened a commercially available library of 1280 compounds (Sigma LOPAC1280) that contains marketed drugs and relevant structures with predictable activities and proven scaffolds directed against a wide range of known drug targets including GPCRs and protein kinases. We screened in duplicate for each cell type at 10 µM in 0.25% DMSO. The robust Z scores for the percent infection are plotted for all 1280 compounds with the replicates on each axis for human cells (Figure 2B) and Drosophila cells (Figure 2C). Drugs with a Z score of <−1.7 in duplicate represent our positive candidates for this screen (p<0.002) and are in the lower left quadrants of Figure 2B–C.


Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

Filone CM, Hanna SL, Caino MC, Bambina S, Doms RW, Cherry S - PLoS ONE (2010)

High-throughput screen for inhibitors of RVFV infection.A. Schematic of screening strategy used in human and insect cells. Cells are seeded in 384 well plates, subsequently 10µM drug was added. The cells were infected with RVFV, then fixed, stained and imaged using automated microscopy. Automated image analysis and statistical thresholds were used to identify inhibitors of RVFV infection. Candidate drugs are then validated and further characterized. B. 6,000 293T cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.06 for 16 hours. Robust Z scores for infection in mammalian were plotted for each replicate in blue. Each of the four plates is denoted by a different symbol. C. 20,000 S2 cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.02 for 48 hours. Robust Z scores for infection in mammalian were plotted for each replicate in red. Each of the four plates is denoted by a different symbol. D. Annotated categories of 59 candidate RVFV inhibitors from LOPAC screen. * denotes over-represented groups p<0.02. E. Venn diagram of the distribution of small molecules: 31 mammalian (blue), 13 insect (red) and 15 pan-inhibitors (purple). * p<10−16. F. Categories of 15 candidates that block infection of both mammalian and Drosophila cells. * denotes over-represented groups p<0.02.
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Related In: Results  -  Collection

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pone-0015483-g002: High-throughput screen for inhibitors of RVFV infection.A. Schematic of screening strategy used in human and insect cells. Cells are seeded in 384 well plates, subsequently 10µM drug was added. The cells were infected with RVFV, then fixed, stained and imaged using automated microscopy. Automated image analysis and statistical thresholds were used to identify inhibitors of RVFV infection. Candidate drugs are then validated and further characterized. B. 6,000 293T cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.06 for 16 hours. Robust Z scores for infection in mammalian were plotted for each replicate in blue. Each of the four plates is denoted by a different symbol. C. 20,000 S2 cells were plated in 384 well plates, treated with inhibitor and infected at MOI = 0.02 for 48 hours. Robust Z scores for infection in mammalian were plotted for each replicate in red. Each of the four plates is denoted by a different symbol. D. Annotated categories of 59 candidate RVFV inhibitors from LOPAC screen. * denotes over-represented groups p<0.02. E. Venn diagram of the distribution of small molecules: 31 mammalian (blue), 13 insect (red) and 15 pan-inhibitors (purple). * p<10−16. F. Categories of 15 candidates that block infection of both mammalian and Drosophila cells. * denotes over-represented groups p<0.02.
Mentions: Next, we used this assay platform to screen for small molecule inhibitors of RVFV MP12 infection both in human (293T) and Drosophila (S2) cells (Schematic Figure 2A). We screened a commercially available library of 1280 compounds (Sigma LOPAC1280) that contains marketed drugs and relevant structures with predictable activities and proven scaffolds directed against a wide range of known drug targets including GPCRs and protein kinases. We screened in duplicate for each cell type at 10 µM in 0.25% DMSO. The robust Z scores for the percent infection are plotted for all 1280 compounds with the replicates on each axis for human cells (Figure 2B) and Drosophila cells (Figure 2C). Drugs with a Z score of <−1.7 in duplicate represent our positive candidates for this screen (p<0.002) and are in the lower left quadrants of Figure 2B–C.

Bottom Line: As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts.Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C.Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

ABSTRACT
As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

Show MeSH
Related in: MedlinePlus