Fig 1.
2DE analysis of CHIKV-infected C6/36 cells.
(A) Twenty-three protein spots of mock-infected samples that showed 2-folds changes with p<0.05 (Student’s t-test) as compared to (B) CHIKV-infected samples were annotated. All the SDS-PAGE gels from the second dimension separation were analyzed using PDQuest 2-D Analysis Software (BioRad). Two biological replicates of the CHIKV-infected samples from each time-point infection were grouped and compared against to the corresponding biological replicates of the mock-infected samples. Representative gels are from 24h mock-infected and 24h CHIKV-infected lysates.
Fig 2.
2-DE dynamic profiles of the selected host proteins upon CHIKV-infection at M.O.I. 10.
Yellow box indicates the differentially expressed proteins. Mock-infection for each time-point served as a control, as shown in panel 24M, 48M and 96M. CHIKV-infection profiles analyzed at 24, 48 and 96 h.p.i., as shown in the panel of 24I, 48I and 96I, respectively. The functional grouping of each protein is shown in UniprotKB database.
Table 1.
Mean change in spot intensities and mass spectrometry data of 23 differentially regulated proteins from CHIKV-infected C6/36 cells at M.O.I. 10.
Fig 3.
STRING analysis of protein-protein interactions between differentially expressed proteins identified through 2DE.
The protein network analysis was performed using STRING v9.05. The interactions between expressed proteins are indicated by the connecting line. The thickness of the connecting line represents the strength of the associations. The interactions network is significantly enriched (p< 0.05).
Fig 4.
Cellular localization and functions of the differentially expressed proteins identified through 2-DE dynamic profiling.
(A) The cellular localizations were determined using UniprotKB database. Most of the identified proteins were found in cytoplasm, and they mainly participate in protein processing. (B) The functions of each protein were determined using KEGG pathways database.
Table 2.
Nine significant KEGG pathways identified in STRING network.
Table 3.
Small interfering (siRNA) sequences used in siRNA based experiments.
Fig 5.
RNA levels on selected cellular genes.
Scrambled siRNA-transfected cells (represented by white bars) against (A) Spermatogenesis-associated factor, (B) Enolase phosphatase e-1 and (C) Chaperonin-60kD show no knockdown of gene expression when compared to transfection control (TC). Cells transfected with targeted cellular siRNAs (represented by shaded bars) against (A) Spermatogenesis-associated factor, (B) Enolase phosphatase e-1 and (C) Chaperonin-60kD showed significant knockdown across all genes tested compared to transfection control (TC). The asterisk indicates *p values <0.05, **p values of <0.01 and ***p values <0.0001 by Student’s t test. Asterisks indicate statistically significant results relative to control group (■).
Fig 6.
siRNA dose-dependent knockdown of Spermatogenesis-associated factor, Enolase phosphatase e-1 and Chaperonin-60kD.
Scrambled siRNAs for (A) Spermatogenesis-associated factor, (C) Enolase phosphatase e-1 and (E) Chaperonin-60kD were subjected to transfection across a range of concentrations (0–50 nM) and then infected with CHIKV. No virus inhibition was observed for any scrambled siRNAs when compared to transfection control (TC). Gene-specific siRNAs against (B) Spermatogenesis-associated factor, (D) Enolase phosphatase e-1 and (F) Chaperonin-60kD were transfected into C6/36 cells at different concentrations (0–50 nM) and subjected to CHIKV infection. Significant dose-dependent inhibition of CHIKV infection was observed in spermatogenesis-associated factor from 30 nM to 50 nM, with approximately 2-log unit PFU/ml reductions. Significant dose-dependent increases in CHIKV titers were observed with enolase phosphatase e-1 and chaperonin-60kD from 10 nM to 50 nM, with approximately 2-log unit PFU/ml increases. Cell viability upon drug treatment is represented by the line graphs. The asterisk indicates *p values <0.05, **p values of <0.01 and ***p values <0.0001 by Student’s t test. Asterisks indicate statistically significant results relative to control group (■).