Fig 1.
Chimeric EIL/VEEV viruses do not replicate in vertebrate cells, and package subgenomic RNAs into viral particles in mosquito cells.
(A) The schematic representation of EILV, VEEV TC-83 and recombinant viral genomes. (B) Replication of EIL/VEEV and EIL/nLuc/VEEV in C7/10 and NIH 3T3 cells. Cells were infected at an MOI of 20 PFU/cell. Media were harvested at the indicated times post infection, and titers were determined by plaque assay on C7/10 cells. (C) Concentrations of genomic RNA, SG RNA 1 and SG RNA 2 in the sample of EIL/GFP/VEEV harvested at 48 h PI of C7/10 cells. The number of copies per ml of virus were determined by RT-qPCR using primers specific to EILV nsP2, GFP and VEEV E2 genes as described in Materials and Methods. (D) C7/10 cells were infected with EIL/GFP/VEEV at an MOI of 20 PFU/cell, and viral RNA were labeled with [3H]uridine between 16 and 24 h post infection. RNAs were isolated from both the cells and the released viral particles and analyzed by agarose gel electrophoresis in denaturing conditions.
Fig 2.
Type I IFN pretreatment inhibits nLuc expression in vertebrate cells in time- and concentration-dependent mode.
(A) The schematic representation of EIL/nLuc/VEEV genome and encoded SG RNAs. (B) NIH 3T3 cells were infected with purified EIL/nLuc/VEEV at an MOI of 10 PFU/cell. After 1 h incubation at 4°C, cells were washed with cold PBS, and then incubated at 37°C in complete media. At the indicated time points, cells were harvested, and nLuc activity was assessed. The control cells were infected and further incubated in the presence of 50 μg/ml puromycin (Pur) and 50 μg/ml cycloheximide (Chx). (C) NIH 3T3 cells were treated for 20 h with IFN-β at the indicated concentrations. Cells were then infected with EIL/nLuc/VEEV at an MOI of 10 PFU/cell for 1 h at 37°C, washed with PBS, and incubated in complete medium at 37°C. nLuc activity was assessed at 4 h post infection. (D) NIH 3T3 cells were treated for indicated times with IFN-β at a concentration of 100 IU/ml. Cells were then infected with EIL/nLuc/VEEV at an MOI of 10 PFU/cell, washed with PBS, and incubated in complete media at 37°C. nLuc activity was analyzed at 4 h post infection. All results are presented as average of triplicate +/- SD in relative luminescence units from a single representative experiment. Most of standard deviations are too small to be seen. The experiments were reproducibly repeated three independent times.
Fig 3.
IFN-β treatment does not affect virus attachment, entry, or disassembly.
(A) NIH 3T3 cells in Ibidi 8-well plates were treated for 20 h with IFN-β at a concentration of 100 IU/ml or mock-treated. Then cells were incubated with VEEV TC-83 (1.5x104 PFU/cell) at 4°C for 1 hour, washed twice with cold PBS and fixed with PFA. Immunostaining of the adsorbed viral particles was performed immediately without any treatment with nonionic detergent using VEEV-specific mouse Abs and AlexaFluor555-labeled secondary Abs. Cell nuclei were stained with Hoechst dye. The three-dimensional images were acquired by confocal microscopy (see Materials and Methods for details), and (B) numbers of bound viral particles per cell were determined using Spot function of the Imaris software. (C) IFN-β-treated or non-treated NIH 3T3 cells were incubated with purified EIL/nLuc/VEEV (MOI of 20 PFU/cell) at 4°C for 1 hour, washed with cold PBS, lysed in Triton X-100-containing buffer, and nLuc activity in the samples was determined. Media from EIL/nLuc/VEEV-infected C710 cells was filtered using 100 kD centrifugal filters to eliminate viral particles and used as a flow-through, virus-free nLuc-containing control. (D) NIH 3T3 cells were treated overnight with 100 IU/ml of IFN-β or left mock-treated. They were incubated with concentrated virus (7.5x103 PFU/cell) at 4°C for 1 h to allow virus interaction with cell surface receptors. Then incubation continued at 37°C for 1 h in complete media in the presence of cycloheximide. Cells were fixed with PFA, permeabilized and stained using rat mAb specific to the amino-terminal fragment of VEEV capsid, and AlexaFluor555-conjugated secondary antibody. Nuclei were stained with Hoechst dye. Images are presented as multiple intensity projections of 6 optical sections. Scale bar is 10 μm.
Fig 4.
Levels of resistance of alphavirus RNA translation to IFN-β is determined by 5’UTRs of their genomes.
(A) The schematic representation of EIL/5’nLuc/VEEV genomes with different 5’UTRs in the nLuc-encoding SG RNAs and the computer predictions (m-Fold) of the RNA foldings. (B) The schematic representation of the EIL/5’EIL-IRES-nLuc/VEEV genome, containing EMCV IRES downstream of the EILV SG RNA-specific 5’UTR. Because of the IRES presence, nLuc is likely translated from both viral genome and SG RNA. (C) wt MEFs were treated for 20 h with different concentrations of IFN-β or remained mock-treated and then infected with the indicated viruses for 1 h at 37°C. The MOI used was adjusted for each virus to obtain similar nLuc activity in the mock-treated samples. After infection, cells were incubated in complete medium at 37°C, and nLuc activity was assessed at 4 h post infection. The results were normalized to the nLuc activity in the samples of mock-treated, infected cells. The experiments were repeated multiple times with reproducible results. Panels represent the data obtained in a single experiment. All of the measurements were performed in triplicates, and standard deviations are too small to be seen.
Fig 5.
IFIT1 inhibits translation of incoming alphavirus-specific RNAs.
(A) Wild type (wt), IFIT1-/-, IFIT2-/- or IFIT locus-/- MEF were treated for 20 h with the indicated concentrations of IFN-β or remained mock-treated. Then cells were infected with EIL/nLuc/VEEV at an MOI of 10 PFU/cell for 1 h at 37°C, washed with PBS, and further incubated in complete medium at 37°C. nLuc activity was analyzed at 4 h post infection, and data were normalized to samples from the mock-treated, infected cells. (B) Wt and IFIT1-/- MEF were treated for 20 h with the indicated concentrations of IFN-β or remained mock-treated. Then cells were infected with EIL/5’wtVEEV-nLuc/VEEV and EIL/5’TCVEEV-nLuc/VEEV viruses. nLuc activity was analyzed at 4 h post infection and normalized to the activity in the samples of the mock-treated, infected cells. One of the reproducible repeated experiments is presented. All of the measurements were performed in triplicates, and standard deviations are too small to be seen.
Fig 6.
IFIT1 is expressed via IFN-dependent and alphavirus replication-dependent pathways.
(A) The schematic representation of VEEV TC-83-based recombinant virus genome (VEEV/GFP/C1) containing mutations in the capsid-specific NLS. (B) The profile of IFIT1 induction in the NIH 3T3 cells treated for 24 h with IFN-β (500 IU/ml) and then incubated in IFN-free media. (C) IFN-α/βR-/- MEFs were infected with VEEV/GFP/C1 at an MOI of 20 PFU/cell. Media were replaced at the indicated time points, and cells were harvested for RNA isolation and analysis. Panels represent titers of the virus and IFIT1 mRNA accumulation profiles. (D) NIH 3T3 cells were infected with VEEV/GFP/C1 at an MOI of 20 PFU/cell. As in the experiment presented in panel (C), media were replaced at the indicated times post infection, and RNAs were isolated from the cells. Panels represent titers of the virus and IFIT1 mRNA accumulation profiles. Two independent RNA samples were prepared for each time point for VEEV/GFP/C1-infected cells and three RNA samples for IFN-β-treated cells.
Fig 7.
IFIT1 inhibits replication of alphaviruses in a concentration-dependent mode.
(A) The schematic representation of the genomes of alphaviruses used in the experiments presented in this figure. (B) Populations of BlaR, IFIT1-expressing and parental cells were used for standard plaque assay of VEEV/GFP and SINV/GFP viruses. Since both viruses were unable to induce plaque formation on stable IFIT1-expressing cells, foci of GFP-expressing cells were detected on a Typhoon phosphorimager. (C) The results of RT-qPCR analysis of IFIT1-specific mRNA in different clones of IFIT1 KI cells, derived from wt MEFs, which were used in the experiments presented on the next panel and in the following sections. (D) The indicated clones of IFIT1 KI cells were infected at an MOI of 1 PFU/cell with VEEV/GFP. Media were replaced at the indicated time points, and virus titers were determined by plaque assay on BHK-21 cells.
Fig 8.
Wild type, but not tissue culture-adapted, alphaviruses are resistant to IFIT1-mediated replication inhibition and induce IFN-β in IFIT1-expressing cells.
(A). Wt MEFs and IFIT1 KI/3 cells were infected with the indicated viruses at an MOI of 5 PFU/cell. Media were harvested at 24 h PI, and cells were stained with Crystal violet at 48 h PI. Virus titers were determined by plaque assay on BHK-21 cells. (B) Concentration of IFN-β was determined in the same harvested samples as described in the Materials and Methods. The experiments were performed two times and in triplicates. Statistical analyses were performed using unpaired T-test. *:p<0.05, **:p<0.01, ***:p<0.001. Means and SD of three experiments are presented
Fig 9.
Alphavirus resistance to IFIT1 can be manipulated genetically and leads to virus attenuation.
(A) The computer prediction of the 5’UTRs of the parental VEEV TC-83 and 5’mutVEEV viruses. To analyze the replication rates, wt MEFs and IFIT1 KI/1 cells were infected at an MOI of 5 PFU/cell with VEEV TC-83 and 5’mutVEEV, media were replaced at the indicated times PI, and virus titers were determined on BHK-21 cells. (B) At 48 h post infection, cells used for analysis of virus replication were stained with crystal violet to assess for CPE. (C) C7/10 cells were infected at an MOI of 10 PFU/cell with VEEV TC-83 and 5’mutVEEV, media were replaced at the indicated times PI, and virus titers were determined on BHK-21 cells. (D) The schematic representation of the CHIKV/GFP 181/25 genome and computer predictions of the original and newly designed 5’UTRs. Rescued viruses were used to infect wt MEFs, IFIT1 KI/2 and IFIT1 KI/3 cell lines at an MOI of 5 PFU/cell, and at 24 h post infection, virus titers were determined by plaque assay on BHK-21 cells. (E and F) Six-day-old NIH Swiss mice were infected via s.c. route with the indicated doses of VEEV TC-83 or the designed VEEV variant with mutated 5’UTR (5’mutVEEV). Mice were monitored daily in terms of weight change (E) and survival (F).Unpaired T-test was used to compare weight change between the VEEV TC-83 and 5’mutVEEV groups (both 2x105 and 106 PFU) at day 4 and 5 post-infection. ***: p<0.001. Survival curves of the VEEV TC-83 and 5’mutVEEV groups (both 2x105 and 106 PFU) are significantly different (p-values <0.001).