Fig 1.
The hypervariable domain of alphavirus nsP3 contains conserved P-rich and FGDF motifs.
(A) Schematic overview of the viruses used in these experiments. Shown are DNA plasmids used as template for the in vitro transcription of chikungunya virus infectious clone (IC) or replicon (rep) RNA. Additionally, the location of deletions and amino acid substitutions used in mutant IC and rep constructs is displayed. (B) Alignment of alphavirus nsP3 amino acid sequences indicates conserved proline rich and FGDF motifs in the HVD. Top: zoom-in on the proline rich region of the nsP3 HVD. Red boxes indicate the PxxPPR motif, while blue boxes indicate the PxPxPR motif. Bottom: Zoom-in on the FGDF motif-containing region of the nsP3 HVD. CHIKV chikungunya virus; BFV Barmah forest virus; SFV Semliki Forest virus; WHAV Whataroa virus; EILV Eilat virus; RRV Ross River virus; MAYV Mayaro virus; BeBV Bebaru virus; TALV Taï Forest alphavirus; EEEV Eastern equine encephalitis virus; VEEV Venezuelan equine encephalitis virus; WEEV Western equine encephalitis virus.
Table 1.
Primers used in this study.
Fig 2.
The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells.
(A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIKrep or CHIKrepΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIKIC, CHIKICP398A or CHIKICPPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID50/ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIKIC, CHIKICP398A or CHIKICPPR401AAA at an MOI of 5 and the TCID50/ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.
Fig 3.
The P-rich motif is not required for transmission of chikungunya virus by Aedes aegypti mosquitoes.
(A) Schematic experimental set-up. Female Ae. aegypti mosquitoes were infected through an infectious bloodmeal containing 1.0 × 107 TCID50/ml of CHIKIC, CHIKICP398A or CHIKICPPR401AAA. At 14 days post infection (dpi) (B) or 7 dpi (C) the infection and transmission rates were determined by infectivity assay on Vero cells. Bars represent cumulative numbers from three (B) or two (C) independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) TCID50/ml of CHIKV in the bodies Ae. aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Vero cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log10 transformed data (α = 0.05).
Fig 4.
At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells.
(A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIKICnsP3mC, CHIKICnsP3mC-FGN, CHIKICnsP3mC-FGC, or CHIKICnsP3mC-FGNC. Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIKICnsP3mC or CHIKICnsP3-FGNC either individually or co-transfected with in vitro transcribed RNA of CHIKIC. Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIKrep or CHIKrep-FGNC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKVIC, CHIKVIC-FGN and CHIKVIC-FGC on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID50/ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKVIC, CHIKVIC-FGN and CHIKVIC-FGC on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID50/ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.
Fig 5.
A single FGDF motif is sufficient for the transmission of chikungunya virus by Aedes aegypti mosquitoes.
(A) Schematic experimental set-up. Female Ae. aegypti mosquitoes were infected through an infectious bloodmeal containing 2.8 × 105 TCID50/ml of CHIKIC, CHIKIC-FGN, CHIKIC-FGC. At 7 days post infection (7dpi) the infection and transmission rates were determined by infectivity assay on (B) Vero and (C) Aag2 cells. Bars represent cumulative numbers from three independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) Ratio between the number of positive bodies or salivas in Vero compared to Aag2 cells. Statistics were performed by Kruskal-Wallis test with Dunn’s post-hoc test (α = 0.05). (E) TCID50/ml in the bodies of Ae. aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Aag2 cells. Statistics was performed by one-way ANOVA with Tukey’s post-hoc test on Log10 transformed data (α = 0.05).
Fig 6.
At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin.
(A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIKICnsP3mC, CHIKICnsP3mC-FGN or CHIKICnsP3mC-FGC. At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIKICnsP3mC, CHIKICnsP3mC-FGN or CHIKICnsP3mC-FGC. At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FGC, nsP3EGFP-FGN or nsP3EGFP-FGNC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FGN, nsP3EGFP-FGC or nsP3EGFP-FGNC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.