Fig 1.
HVD of VEEV nsP3 binds proteins of FXR family.
(A) Schematic presentation of replicons expressing GFP-HVDveev and GFP-HVDsinv fusion proteins. (B) List of proteins co-isolated with GFP-HVDveev and GFP-HVDsinv from BHK-21 cells. Total spectra for 2 independent immunoprecipitation (IP) experiments are presented. (C) Colocalization of VEEV nsP3 with FXR proteins at 6 h PI at an MOI of 20. Pearson's colocalization coefficients are shown in first overlay panels (mean±SD, n cells >12). Scale bars: 20 μm for 2 left panels or 3 μm for 3 right panels.
Fig 2.
The knockout of Fxr or G3bp genes differentially affects replication of alphaviruses.
(A) Left panels: growth curves of the indicated alphaviruses in NIH 3T3, Fxr dKO and Fxr tKO cells (MOI 1). Right panels: bar graphs show titers of the indicated viruses in NIH 3T3 and Fxr tKO cells at 8 h PI (MOI 0.05, three biological repeats). (B) Left panels: growth curves of the indicated alphaviruses in NIH 3T3, G3bp2 KO and G3bp dKO cells (MOI 0.01). Right panels: bar graphs show titers of the indicated viruses in NIH 3T3 and G3bp dKO cells at 8 h PI (MOI 0.05, three biological repeats). (C, D) Sizes of plaques developed in NIH 3T3/Fxr tKO (C) or NIH 3T3/G3bp dKO (D) cells by the indicated viruses. Due to strong reduction in infectivity of KO cells wells with different virus dilutions are shown. Data in bar graphs shown in (A) and (B) are mean±SD of 3 biological repeats. DL indicates a detection limit.
Fig 3.
Any single FXR or G3BP protein supports virus replication.
(A, B) A single, ectopically expressed FXR or G3BP protein supports replication of VEEV and CHIKV, respectively. Single FXR or G3BP-GFP proteins were stably expressed in Fxr tKO or G3bp dKO cells, respectively. VEEV titers were determined at 8 h PI at an MOI 0.05. CHIKV titers were determined at 7 h PI at an MOI of 0.1. (C, D) Analysis of function of FXR1 and G3BP1 domain deletion mutants in VEEV and CHIKV replication, respectively. The experiments were performed in stable cell lines, established in Fxr tKO or G3bp dKO cells. VEEV titers were assessed at 8 h PI (MOI 0.05), and CHIKV titers were determined at 7 h PI (MOI 0.1). n.d. indicates that virus replication was below the detection limit. n.a. indicates that this cell line was not available. Data are mean±SD of 3 biological repeats.
Fig 4.
FXRs and G3BPs facilitate vRC formation and RNA replication.
(A) FACS analysis of NIH 3T3, Fxr tKO and G3bp dKO cells, infected with VEEV/GFP and CHIKV/GFP (4 h PI, MOI 10). (B) Replication rates of a chimeric virus VEE/CHIKV in the indicated cells (MOI of 0.1). (C) Translation of nLuc-encoding RNA with alphavirus genome-specific 5’UTRs in the indicated cell lines. Data are mean±SD of 3 biological repeats. Deviations are too small to be visible in the used log scale. (D) Accumulation of VEEV- and CHIKV-specific G RNAs, measured by RT-qPCR, in the indicated cells infected at an MOI of 10 (mean±SD, 3 repeats). (E) Accumulation of nsP3 and capsid protein in Fxr dKO, Fxr tKO cells and parental NIH 3T3 cells. Cells were infected with VEEV TC-83 at an MOI of 20, harvested at different times PI, and the lysates were analyzed by Western blot using nsP3-specific and capsid-specific Abs. Quantitative analysis was performed on LI-COR imager. n.d. indicates that protein concentration was below the detection level. (F) Numbers of vRCs per cell at different times PI with VEEV TC-83. The indicated p values are between NIH 3T3 and KO cells at the same time points. (G) Numbers of vRCs in indicated cells at 3 h PI with VEEV TC-83. The p values in black are between NIH 3T3 and KI cell lines, the p values in blue are between Fxr tKO and KI cells. Data in F and G are presented as median with interquartile range. The p values were estimated using Mann-Whitney test; n cells per group > 29 (see Materials and Methods for details).
Fig 5.
FXR-nsP3 complexes bind viral G RNA in VEEV-infected cells.
(A) Colocalization of VEEV nsP3-GFP fusion proteins with VEEV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane on nucleus. Bars: 10 and 3 μm. (B) Colocalization of FXR1-GFP with VEEV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane of the nucleus. Bars: 10 and 3 μm. (C) Localization of VEEV nsP3-GFP, FXR1 and dsRNA at the plasma membrane at 2 h PI. Images are presented as MIP of x-z sections (1 μm). Bars: 10 μm. (D) The x-y 1-μm-thick section at the plasma membrane of the cell presented in C. The dsRNAs are presented as spots rendered in Imaris software. Bars: 3 and 1 μm. (E) Localization of VEEV nsP3-GFP, dsRNA and G RNA at the plasma membrane at 2 h PI. Images are presented as MIP of x-y 1-μm-thick sections at the plasma membrane. The dsRNAs are presented as spots rendered in Imaris. Bars: 10 and 3 μm. Yellow arrowheads indicate colocalization of nsP3-GFP and G RNA in A or nsP3-GFP and FXR1 in D. Red arrowheads indicate G RNA spots lacking nsP3-GFP. Green arrowheads indicate positions of nsP3-GFP complexes, containing no G RNA. Turquoise arrowheads indicate fully formed, dsRNA-positive vRCs, which contain low levels of G3BP1-GFP. Pink arrowheads indicate the dsRNA signals of low intensity and volume, which is suggesting the presence of shorter dsRNAs.
Fig 6.
G3BP-nsP3 complexes bind viral G RNA in CHIKV-infected cells.
(A) Colocalization of CHIKV nsP3-Cherry (pseudocolored in green) fusion proteins with CHIKV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane of nucleus. Bars: 10 and 3 μm. (B) Colocalization of CHIKV nsP3 with G3BP2, stained with specific Abs at the plasma membrane at 2 h PI. Bars: 10 μm and 3 μm. (C) At 2 h PI, G3BP2 and dsRNA form stripes at the plasma membrane in CHIKV-infected cells. Staining was performed with specific Abs. (D) CHIKV G RNA and G3BP1-GFP strongly colocalize at the plasma membrane in the infected cells. The dsRNA-containing vRCs are formed in close proximity to G3BP-GFP/G RNA complexes and often overlap. Bars: 2 μm. Images in B-D are presented as MIP of x-y sections (1 μm) at the plasma membrane. Yellow arrowheads indicate pre-vRCs, which appear as dsRNA-free G3BP2-positive spots (red) in (C) or G RNA/G3BP1-GFP spots (yellow) in (D). Turquoise arrowheads indicate fully formed, dsRNA-positive vRCs, which contain low levels of G3BP1-GFP. Pink arrowheads indicate pre-vRCs, which contain a dsRNA signal of low intensity and volume and, likely, represent vRCs in the process of synthesis of dsRNA intermediate.
Fig 7.
Repeating aa sequences in VEEV and CHIKV HVDs determine virus replication.
(A) The schematic presentation of the recombinant VEEV genome and modifications introduced into nsP3 HVD sequence. Replacement of repeating sequences in VEEV HVD by those derived from CHIKV HVD makes VEEV/chikvR/GFP replication dependent on G3BP. VEEV/mutHVD/GFP titers were determined at 7 h PI, and VEEV/chikvR/GFP titers were determined at 8 h PI (MOI 0.01). (B) The schematic presentation of the recombinant CHIKV genome and modifications introduced into nsP3 HVD sequence. SINV-specific sequence in CHIKV HVD, but not VEEV repeating element, can support CHIKV replication. Virus titers were determined at 24 h PI (MOI of 0.05). Data in (A) and (B) are presented as mean±SD of 3 biological repeats. NV indicates that the designed mutants were not viable. (C) Colocalization of wt and mutant VEEV nsP3 proteins with FXR1 and G3BP2. Images are presented as MIP of x-y 1-μm-thick sections at the plasma membrane. Pearson's colocalization coefficients are shown in overlay panel (mean±SD, n>6). Bars: 2 μm
Fig 8.
Model of FXR- and G3BP-mediated alphavirus replication complex formation.
Binding of FXRs or G3BPs to the PM-bound P123 and P123+nsP4 complexes promotes their interaction with newly synthesized G RNAs and formation of pre-vRCs. In the absence of FXRs or G3BPs, G RNAs are acquired by P123+nsP4 complexes less efficiently and are likely degraded.