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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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Time-of-Addition Assays.A. 293T/17 cells were either pre-treated with the indicated drugs or treated with drugs 1 hour post infection (MOI = 0.3) for 12–15 hours. Representative images from cells treated with NH4Cl, Ribavirin, EIPA, Rottlerin and Vinblastine are shown. Cells stained with FITC anti-RVFV N (green) and counterstained with DAPI (blue). B. Quantification of the time of addition assay in A. was graphed to show the normalized percent infection for the indicated drugs tested. Blue bars are the pre-treatment, and red bars are the one hour post infection treatments.
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pone-0015483-g004: Time-of-Addition Assays.A. 293T/17 cells were either pre-treated with the indicated drugs or treated with drugs 1 hour post infection (MOI = 0.3) for 12–15 hours. Representative images from cells treated with NH4Cl, Ribavirin, EIPA, Rottlerin and Vinblastine are shown. Cells stained with FITC anti-RVFV N (green) and counterstained with DAPI (blue). B. Quantification of the time of addition assay in A. was graphed to show the normalized percent infection for the indicated drugs tested. Blue bars are the pre-treatment, and red bars are the one hour post infection treatments.

Mentions: The lifecycle of RVFV, like that of any virus, involves sequential steps that can be kinetically dissected. Performing time-of-addition experiments can determine if an inhibitor blocks early versus late steps in the lifecycle. We found that binding and entry of RVFV MP12 takes approximately one hour in 293T cells because treatment with lysosomotropic agents such as Ammonium Chloride one hour post-infection no longer blocked infection (Figure 4). In contrast, we found that the nucleoside analog Ribavirin, which inhibits RNA replication, remained inhibitory when added one hour post-infection (Figure 4). Therefore, by treating cells with inhibitors at one hour post-infection, we could determine whether the small molecules targeted an entry or post-entry step in the RVFV MP12 lifecycle.


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)

Time-of-Addition Assays.A. 293T/17 cells were either pre-treated with the indicated drugs or treated with drugs 1 hour post infection (MOI = 0.3) for 12–15 hours. Representative images from cells treated with NH4Cl, Ribavirin, EIPA, Rottlerin and Vinblastine are shown. Cells stained with FITC anti-RVFV N (green) and counterstained with DAPI (blue). B. Quantification of the time of addition assay in A. was graphed to show the normalized percent infection for the indicated drugs tested. Blue bars are the pre-treatment, and red bars are the one hour post infection treatments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015483-g004: Time-of-Addition Assays.A. 293T/17 cells were either pre-treated with the indicated drugs or treated with drugs 1 hour post infection (MOI = 0.3) for 12–15 hours. Representative images from cells treated with NH4Cl, Ribavirin, EIPA, Rottlerin and Vinblastine are shown. Cells stained with FITC anti-RVFV N (green) and counterstained with DAPI (blue). B. Quantification of the time of addition assay in A. was graphed to show the normalized percent infection for the indicated drugs tested. Blue bars are the pre-treatment, and red bars are the one hour post infection treatments.
Mentions: The lifecycle of RVFV, like that of any virus, involves sequential steps that can be kinetically dissected. Performing time-of-addition experiments can determine if an inhibitor blocks early versus late steps in the lifecycle. We found that binding and entry of RVFV MP12 takes approximately one hour in 293T cells because treatment with lysosomotropic agents such as Ammonium Chloride one hour post-infection no longer blocked infection (Figure 4). In contrast, we found that the nucleoside analog Ribavirin, which inhibits RNA replication, remained inhibitory when added one hour post-infection (Figure 4). Therefore, by treating cells with inhibitors at one hour post-infection, we could determine whether the small molecules targeted an entry or post-entry step in the RVFV MP12 lifecycle.

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