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Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex.

Reid SP, Tritsch SR, Kota K, Chiang CY, Dong L, Kenny T, Brueggemann EE, Ward MD, Cazares LH, Bavari S - Emerg Microbes Infect (2015)

Bottom Line: Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness.SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins.Collectively these results identify SK2 as a novel CHIKV host factor.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Translational Science, US Army Medical Research Institute of Infectious Diseases (USAMRIID) , Frederick, MD 21702-5011, USA.

ABSTRACT
Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.

No MeSH data available.


Related in: MedlinePlus

SK2 inhibition impairs CHIKV infection. (A and B) HepG2 cells were pretreated for 2 h with 5, 10, 20, or 30 µM of SKI-II (A) or ABC (B) then infected with CHIKV at an MOI of 5. At 48-h postinfection, cells were fixed and analyzed for infection by high-content confocal microscopy. Values represent mean + SEM (n = 3). *P < 0.05, **P < 0.01 (C) HeLa cells were pretreated with either 50, 100, or 500 nM S1P for 15 min then infected with CHIKV at a MOI if 5 for 24 h. Cell lysates were harvested and subjected to Western blot analysis to detect the E2 viral glycoprotein and GAPDH, the relative intensity for each band is shown below.
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fig2: SK2 inhibition impairs CHIKV infection. (A and B) HepG2 cells were pretreated for 2 h with 5, 10, 20, or 30 µM of SKI-II (A) or ABC (B) then infected with CHIKV at an MOI of 5. At 48-h postinfection, cells were fixed and analyzed for infection by high-content confocal microscopy. Values represent mean + SEM (n = 3). *P < 0.05, **P < 0.01 (C) HeLa cells were pretreated with either 50, 100, or 500 nM S1P for 15 min then infected with CHIKV at a MOI if 5 for 24 h. Cell lysates were harvested and subjected to Western blot analysis to detect the E2 viral glycoprotein and GAPDH, the relative intensity for each band is shown below.

Mentions: To chemically confirm the results of the previous experiments, we utilized the well-characterized SK-specific inhibitor, sphingosine kinase inhibitor II (SKI-II).38,39 HepG2 cells were pretreated with increasing concentrations of SKI-II (5, 10, 20, 30 µM), then subsequently infected. Treatment with SKI-II (between 20 µM and 30 µM) resulted in a significant reduction in CHIKV infection (Figure 2A). It is interesting to note, SKI-II was similarly shown to inhibit Influenza A infection at 30 µM.25 SKI-II is a dual SK inhibitor able to inhibit both SK1 and SK2.40 Because CHIKV infection was shown to be dependent on SK2 it was of interest to use a SK2-specific inhibitor. For this, we used the SK2-specific inhibitor ABC294640 (ABC).40,41 HepG2 cells were pretreated with increasing concentrations of ABC then infected with virus. ABC pretreatment potently inhibited CHIKV infection in a dose-dependent manner (Figure 2B). Comparatively, virus inhibition by ABC at 10 µM was greater than that observed for SKI-II at the same concentration. Since the enzymatic function of SK2 similar to SK1 is to catalyze the production of S1P from sphingosine, and our data thus far indicate SK2 is positively required for CHIKV infection, we wanted to determine if treatment with exogenous S1P would similarly impact virus infection. Pretreatment with increasing concentration of S1P had only a modest effect on viral protein expression (Figure 2C). These results demonstrate that targeting SK2 significantly inhibits CHIKV infection, while targeting the S1P system by the addition of exogenous S1P did not appear to affect viral protein production.


Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex.

Reid SP, Tritsch SR, Kota K, Chiang CY, Dong L, Kenny T, Brueggemann EE, Ward MD, Cazares LH, Bavari S - Emerg Microbes Infect (2015)

SK2 inhibition impairs CHIKV infection. (A and B) HepG2 cells were pretreated for 2 h with 5, 10, 20, or 30 µM of SKI-II (A) or ABC (B) then infected with CHIKV at an MOI of 5. At 48-h postinfection, cells were fixed and analyzed for infection by high-content confocal microscopy. Values represent mean + SEM (n = 3). *P < 0.05, **P < 0.01 (C) HeLa cells were pretreated with either 50, 100, or 500 nM S1P for 15 min then infected with CHIKV at a MOI if 5 for 24 h. Cell lysates were harvested and subjected to Western blot analysis to detect the E2 viral glycoprotein and GAPDH, the relative intensity for each band is shown below.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631929&req=5

fig2: SK2 inhibition impairs CHIKV infection. (A and B) HepG2 cells were pretreated for 2 h with 5, 10, 20, or 30 µM of SKI-II (A) or ABC (B) then infected with CHIKV at an MOI of 5. At 48-h postinfection, cells were fixed and analyzed for infection by high-content confocal microscopy. Values represent mean + SEM (n = 3). *P < 0.05, **P < 0.01 (C) HeLa cells were pretreated with either 50, 100, or 500 nM S1P for 15 min then infected with CHIKV at a MOI if 5 for 24 h. Cell lysates were harvested and subjected to Western blot analysis to detect the E2 viral glycoprotein and GAPDH, the relative intensity for each band is shown below.
Mentions: To chemically confirm the results of the previous experiments, we utilized the well-characterized SK-specific inhibitor, sphingosine kinase inhibitor II (SKI-II).38,39 HepG2 cells were pretreated with increasing concentrations of SKI-II (5, 10, 20, 30 µM), then subsequently infected. Treatment with SKI-II (between 20 µM and 30 µM) resulted in a significant reduction in CHIKV infection (Figure 2A). It is interesting to note, SKI-II was similarly shown to inhibit Influenza A infection at 30 µM.25 SKI-II is a dual SK inhibitor able to inhibit both SK1 and SK2.40 Because CHIKV infection was shown to be dependent on SK2 it was of interest to use a SK2-specific inhibitor. For this, we used the SK2-specific inhibitor ABC294640 (ABC).40,41 HepG2 cells were pretreated with increasing concentrations of ABC then infected with virus. ABC pretreatment potently inhibited CHIKV infection in a dose-dependent manner (Figure 2B). Comparatively, virus inhibition by ABC at 10 µM was greater than that observed for SKI-II at the same concentration. Since the enzymatic function of SK2 similar to SK1 is to catalyze the production of S1P from sphingosine, and our data thus far indicate SK2 is positively required for CHIKV infection, we wanted to determine if treatment with exogenous S1P would similarly impact virus infection. Pretreatment with increasing concentration of S1P had only a modest effect on viral protein expression (Figure 2C). These results demonstrate that targeting SK2 significantly inhibits CHIKV infection, while targeting the S1P system by the addition of exogenous S1P did not appear to affect viral protein production.

Bottom Line: Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness.SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins.Collectively these results identify SK2 as a novel CHIKV host factor.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Translational Science, US Army Medical Research Institute of Infectious Diseases (USAMRIID) , Frederick, MD 21702-5011, USA.

ABSTRACT
Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.

No MeSH data available.


Related in: MedlinePlus