Limits...
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.

Show MeSH

Related in: MedlinePlus

PKCepsilon is required for RVFV infection of human cells.A. Human 293T cells were transfected with siRNAs against a control, or the novel PKC isozymes PKCδ or PKCε. The depleted cells were challenged with RVFV MP12 (MOI = 1) for 12 hours were processed for immunofluoresecnce and quantified. Mean±sd for three experiments; * p<0.02. B. PKCε is depleted by siRNA treatment as measured by immunoblot compared to a non-specific control (NS). C. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with RVFV MP12 for plaque assays in duplicate for each experiment, averaged, and the fold-change compared to control of the pfu/mL was averaged across three independent experiments; * p<0.02. D. PKCε was depleted by RNAi as measured by immunoblot compared to a tubulin control. E. Human 293T cells were transfected with siRNAs against a control, or the PKCε and the depleted cells were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments. F. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991366&req=5

pone-0015483-g005: PKCepsilon is required for RVFV infection of human cells.A. Human 293T cells were transfected with siRNAs against a control, or the novel PKC isozymes PKCδ or PKCε. The depleted cells were challenged with RVFV MP12 (MOI = 1) for 12 hours were processed for immunofluoresecnce and quantified. Mean±sd for three experiments; * p<0.02. B. PKCε is depleted by siRNA treatment as measured by immunoblot compared to a non-specific control (NS). C. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with RVFV MP12 for plaque assays in duplicate for each experiment, averaged, and the fold-change compared to control of the pfu/mL was averaged across three independent experiments; * p<0.02. D. PKCε was depleted by RNAi as measured by immunoblot compared to a tubulin control. E. Human 293T cells were transfected with siRNAs against a control, or the PKCε and the depleted cells were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments. F. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments.

Mentions: Because small molecules may inhibit more than one protein, thus having off target effects, we wanted to validate the inhibitor results using an independent approach. To this end, we used RNA interference technology to specifically deplete each of the two major nPKC isozymes, PKCdelta (PKCδ) and PKCepsilon (PKCε). We transiently transfected 293T cells with either a control non-targeting siRNA or a siRNA against PKCδ or PKCε, waited three days for depletion, and challenged the cells with RVFV MP12 for 12 hours. Under these conditions we consistently observed an approximately two-fold decrease in the percentage of infected cells that were depleted for PKCε compared to both the controls and PKCδ-depleted cells at two different MOIs (Figure 5A). Immunoblot analyses demonstrated that the siRNA against PKCε significantly depleted, but did not eliminate, PKCε protein levels compared to untreated or non-targeting controls (Figure 5B).


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)

PKCepsilon is required for RVFV infection of human cells.A. Human 293T cells were transfected with siRNAs against a control, or the novel PKC isozymes PKCδ or PKCε. The depleted cells were challenged with RVFV MP12 (MOI = 1) for 12 hours were processed for immunofluoresecnce and quantified. Mean±sd for three experiments; * p<0.02. B. PKCε is depleted by siRNA treatment as measured by immunoblot compared to a non-specific control (NS). C. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with RVFV MP12 for plaque assays in duplicate for each experiment, averaged, and the fold-change compared to control of the pfu/mL was averaged across three independent experiments; * p<0.02. D. PKCε was depleted by RNAi as measured by immunoblot compared to a tubulin control. E. Human 293T cells were transfected with siRNAs against a control, or the PKCε and the depleted cells were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments. F. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991366&req=5

pone-0015483-g005: PKCepsilon is required for RVFV infection of human cells.A. Human 293T cells were transfected with siRNAs against a control, or the novel PKC isozymes PKCδ or PKCε. The depleted cells were challenged with RVFV MP12 (MOI = 1) for 12 hours were processed for immunofluoresecnce and quantified. Mean±sd for three experiments; * p<0.02. B. PKCε is depleted by siRNA treatment as measured by immunoblot compared to a non-specific control (NS). C. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with RVFV MP12 for plaque assays in duplicate for each experiment, averaged, and the fold-change compared to control of the pfu/mL was averaged across three independent experiments; * p<0.02. D. PKCε was depleted by RNAi as measured by immunoblot compared to a tubulin control. E. Human 293T cells were transfected with siRNAs against a control, or the PKCε and the depleted cells were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments. F. Human H358 cells stably expressing either a control hairpin or a PKCε specific hairpin were challenged with poliovirus for 8 hours, processed for immunofluorescence and quantified. Mean±sd for three experiments.
Mentions: Because small molecules may inhibit more than one protein, thus having off target effects, we wanted to validate the inhibitor results using an independent approach. To this end, we used RNA interference technology to specifically deplete each of the two major nPKC isozymes, PKCdelta (PKCδ) and PKCepsilon (PKCε). We transiently transfected 293T cells with either a control non-targeting siRNA or a siRNA against PKCδ or PKCε, waited three days for depletion, and challenged the cells with RVFV MP12 for 12 hours. Under these conditions we consistently observed an approximately two-fold decrease in the percentage of infected cells that were depleted for PKCε compared to both the controls and PKCδ-depleted cells at two different MOIs (Figure 5A). Immunoblot analyses demonstrated that the siRNA against PKCε significantly depleted, but did not eliminate, PKCε protein levels compared to untreated or non-targeting controls (Figure 5B).

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