Figure 1.
Determination of the cytotoxicity of Hsp90 inhibitor, Geldanamycin (GA).
(a)Vero cells were treated with different concentrations of GA (1, 5, 25, 50, 100, 200, 500 µM) for 14 h and the cytotoxicity of the cells were determined by MTT assay. Cellular cytotoxicity was determined in duplicate and each experiment was repeated three times. (b) Cells were observed under microscope (Magnification -10X) for cytotoxicity with increasing concentration of the drug (10, 50, 100, 200 µM) on Vero cells at 14 h. (c) Vero cells treated with (10, 50, 100, 200 µM) of GA were harvested at 24 h, lysed and expression of Hsp90 was analysed in Western blot by probing with Hsp90 antibody. GAPDH served as the loading control. The changes in the band intensity was quantified by normalizing GAPDH and the relative band intensity has been shown as bar diagram in right panel (n = 3;p<0.05).
Figure 2.
Hsp90 inhibitor GA inhibits CHIKV replication.
(a) Vero cells were infected with CHIKV strains, either S 27 or DRDE-06 with MOI 0.01. Effect of GA (10, 50, 100, 200 µM) on (a) uninfected cells (b) in the progression of infection was determined by observing the CPE of the virus infected cells under microscope (Magnification -10X) at 18 and 24 hpi. (c) The mock and CHIKV (S 27 or DRDE-06) infected Vero cells in presence or absence of GA (50 µM) were analyzed for apoptosis by staining with Annexin V and estimating % positive cells for Annexin V. The graph depicts a representative experiment with triplicate values of mean ±SD (*p<0.05). (d) The supernatants of the virus infected and GA treated cells were collected at 14 hpi and viral titers were determined by plaque assay. The data represent the mean ±SD of three experiments (*p<0.01). (e) GA was added to the virus infected cells at 0, 4, 6, 8 hpi and supernatants were collected at 14 hpi and infectious progeny virus particle titre was determined by plaque assay. All the experiments were repeated three times and data of three independent experiments are represented as mean ±SD (*p<0.05). (f) The infected cells (as mentioned in 2e) were harvested and nsP2 protein level was determined by Western blot by probing with nsP2 monoclonal antibody. GAPDH served as the loading control. The fold change in the nsP2 protein level was quantified by normalizing GAPDH and the relative band intensity is shown as bar diagram in the lower panel (n = 3, p<0.01).
Figure 3.
Effect of GA on virus replication with different MOIs of CHIKV.
Vero cells were infected with different MOIs (1, 0.1, 0.01) of either S 27 or DRDE-06 virus and treated with 50 µM concentration of GA. (a) The cells were harvested at 8 hpi, lysed and Western blot analysis was performed by probing with nsP2 monoclonal antibody. GAPDH served as the loading control. The reduction in nsP2 level was quantified and the relative band intensity is shown as bar diagram in the right panel (n = 3, p<0.05) (b) and (c) The supernatants were collected at 8 hpi and infective progeny virus particle titre was determined by Plaque assay. Data of three independent experiments are represented as mean ±SD (*p<0.05).
Figure 4.
GA reduces nsP2 protein level during CHIKV infection.
Vero cells were infected with either S 27 or DRDE-06 with MOI 0.01 of the virus. (a) Cells were treated with different doses (10 and 50 µM) of GA and the virus infected cells were harvested at 14 hpi and RT- PCR was performed to amplify nsP1, nsP2, nsP3, nsP4, Hsp90AA1 and GAPDH genes.(b) Virus infected cells were treated with 10, 50 and 100 µM doses of GA and harvested at 14 hpi. Western blot analysis was performed with cell lysates and probed with nsP1, nsP2, nsP3, nsP4 and Hsp90 antibody. GAPDH served as the loading control. The band intensities of nsP1-4 were measured for DMSO and 10 µM GA treated samples after normalizing GAPDH and error bars represent the S.D. of the data from three independent experiments (* p<0.01). (c) Vero cells were infected with either S 27 or DRDE-06 and treated with 50 µM GA. Cells were harvested at 0, 4, 8 and 12 hpi and probed with nsP2 monoclonal antibody. (d) Vero cells were mock transfected or transfected with 10, 30 and 60 pmol of HSP90AA1 gene siRNA. Hsp90 level was estimated in Western blot by probing with Hsp90 antibody (upper panel). After 24 hrt (30 pmol), cells were super infected with MOI1 of either S 27 or DRDE-06 and harvested at 8 hpi. nsP2 protein level was analysed by Western blot (lower panel). The changes in Hsp90 and nsP2 level were quantified by normalizing GAPDH and the relative band intensity is shown as bar diagram in the right panel (n = 3; p<0.05). (e) Vero cells were infected with either S 27 or DRDE-06 (MOI 0.1) virus in presence or absence of GA (50 µM). The cell lysates harvested at 10 and12 hpi for DRDE-06 and S 27 respectively, were co-immunoprecipitated with either polyclonal nsP2 or monoclonal Hsp90 antibodies. Western blot analysis was performed to check the interaction between nsP2 with Hsp90. The extreme right panel represents the negative control where normal mouse IgG was used to pull down the protein complex.
Figure 5.
Reduction in nsP2 level and virus titer when cells were serum starved.
Vero cells were serum starved for 48(a) Vero cells were infected with S 27 virus and treated with different concentrations of the drug (1, 5, 10, 20 µM) and cells were harvested at 14 hpi. Western blot was performed with cell lysates and probed with nsP2 antibody (b) Vero cells were infected either with S 27 or DRDE-06 and drug treated supernatants were collected at 14 hpi. Viral titre was determined by plaque assay. The data represents the mean ±SD with three independent experiments (*p<0.05). (c) Vero cells were infected with either S 27 or DRDE-06 and the drug (1 µM) was added during infection as well as after infection and the supernatants were collected at 14 hpi and plaque assay was performed to determine virus titre. Data of three independent experiments are represented as mean ±SD (*p<0.05). (d) The cells were harvested from the virus infected dishes as mentioned in 5(c) and Western blot was performed using the cell lysates collected from serum starved and nutrient rich condition (with and without 1 µM GA) and was probed for nsP2 and GAPDH antibody. A = addition of GA after infection. D+A = addition of drug during as well as after infection.
Figure 6.
Enhanced activation of Hsp client proteins after CHIKV infection.
Vero cells were infected with either S 27 or DRDE-06 with MOI 0.1 and treated with different doses of GA (10 and 50 µM) and the cells were harvested at 8hpi. Western blot was performed using cell lysates and probed with Hsp90, Raf1, Ras, Akt, pAkt, GSK3β, mTOR, pmTOR, S6K, p70S6K, 4EBP1, p4EBP1 and nsP2 antibodies. GAPDH was used as the loading control.
Figure 7.
Schematic representation of the working model of the cellular signalling molecules in CHIKV infection.
CHIKV infection results in the induction of Hsp90 associated client proteins like Raf1, Akt and various Akt substrates. GA treatment on CHIKV infected cells results in the degradation of Hsp90 associated client proteins which has been explained in the text.