Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode

Abstract

Alphaviruses are a family of positive-strand RNA viruses that circulate on all continents between mosquito vectors and vertebrate hosts. Despite a significant public health threat, their biology is not sufficiently investigated, and the mechanisms of alphavirus replication and virus-host interaction are insufficiently understood. In this study, we have applied a variety of experimental systems to further understand the mechanism by which infected cells detect replicating alphaviruses. Our new data strongly suggest that activation of the antiviral response by alphavirus-infected cells is determined by the integrity of viral genes encoding proteins with nuclear functions, and by the presence of two cellular pattern recognition receptors (PRRs), RIG-I and MDA5. No type I IFN response is induced in their absence. The presence of either of these PRRs is sufficient for detecting virus replication. However, type I IFN activation in response to pathogenic alphaviruses depends on the basal levels of RIG-I or MDA5.

Keywords: Alphaviruses; Innate immunity; MDA5; Pattern recognition receptors; RIG-I; Type I interferon; Virus–host interactions.

Fig. 1

Alphaviruses deficient in nuclear functions, but not their wt variants, induce high levels of IFN-β. (A) The schematic representation of alphavirus genomes encoding wt and mutated nsP2- and capsid-coding genes (SINV/G/GFP and VEEV/GFP/Cm, respectively). (B) NIH 3T3 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. Media were harvested at 16 h post infection and used for assessment of both virus titers and IFN-β levels. Means of three biological repeats with SD are presented.

Fig. 2

RIG-I and MDA5 expression is strongly inhibited in the selected KD cell lines. (A) Equal numbers of NIH 3T3 cells and indicated clone-derived cell lines were treated with mouse IFN-β (500 IU/ml) for 24 h or remained untreated. Cell lysates, corresponding to equal numbers of cells were analyzed by SDS-PAGE, followed by Western blot using MDA5- and RIG-I-specific, and tubulin-specific antibodies in conjunction with infrared dye-labeled secondary antibodies. Membranes were scanned on a LI-COR imager. (B) The indicated clone-derived cell lines were treated with mouse IFN-β (500 IU/ml) for 24 h. RNAs were isolated, and relative concentrations of RIG-I- and MDA5-specific mRNAs were determined by RT-qPCR as described in the Materials and Methods.

Fig. 3

Single PRR KD, but not dKD, cells respond to alphavirus replication by IFN-β release. (A) Parental NIH 3T3, KD RIG-I, KD MDA5 and dKD cells were infected with SINV/G/GFP and VEEV/GFP/Cm viruses at an MOI of 20 PFU/cell. Media was replaced at 24 h post infection and harvested again at 48 h post infection. Concentration of IFN-β was assessed as described in the Materials and methods. Sensitivity of the assay was ~1 pg/ml. Means of three biological repeats with SD are presented. (B) NIH 3T3 and dKD cells were transfected with poly(I:C) using the indicated transfection reagents or treated with poly(I:C)-containing media as described in the Materials and Methods. Media were harvested at 18 h post transfection, and concentration of IFN-β was determined as indicated above. This experiment was repeated twice with reproducible results. (C) NIH 3T3 and dKD cells were infected with SINV/G/GFP and VEEV/GFP/Cm viruses at an MOI of 20 PFU/cell for 8 h or treated with IFN-β at a concentration of 500 IU/ml for 30 min. Cell lysates were analyzed by Western blot using p-STAT1-, STAT1-, GFP- and β-tubulin-specific antibodies.

Fig. 4

Single PRR KD cells are capable of both downregulating the spread of mutant alphaviruses and inhibiting already established virus replication. (A) NIH 3T3, KD RIG-I, KD MDA5 and dKD cells were seeded into 6-well Costar plates at a concentration of 5×10⁵ cells per well. SINV/G/GFP and VEEV/GFP/Cm virus stocks were serially diluted and used for infection of the indicated cells with different numbers of infectious units. After 1-h-long incubation at 37°C, the virus-containing media were replaced with 2 ml of media supplemented with 0.6% agarose. After incubation at 37°C for 48 h, cells were fixed, and plates were scanned on a Typhoon imager to enumerate and assess foci of GFP-expressing cells. Images represent wells infected with the same numbers of infectious units. (B) Subconfluent monolayers of NIH 3T3, KD RIG-I, KD MDA5 and dKD cells were infected with SINV/G/GFP and VEEV/GFP/Cm at an MOI of 20 PFU/cell. Media were replaced every day and cells were split upon reaching confluency, usually every 24 hours. Virus titers in harvested media were determined by plaque assay on BHK-21 cells. Dashed lines represent the limits of detection. This experiment was repeated twice with similar results.

Fig. 5

Single KD and dKD cells remain fully capable of responding to IFN-β treatment and activating the antiviral state. (A) NIH 3T3, KD RIG-I and dKD cells were treated with IFN-β at a concentration of 500 IU/ml for 20 h or mock-treated and then infected with SINV/GFP at an MOI of 5 PFU/cell. Media were replaced at the indicated time points and virus titers were determined by plaque assay on BHK-21 cells. (B) dKD cells were infected with the VEEV/GFP/Cm mutant at an MOI of 5 PFU/cell and incubated for 5 days with periodic splitting. After 5 days, the persistently infected cells were either treated with IFN-β at concentration of 1000 IU/ml or mock treated. Titers of released virus were assessed by plaque assay on BHK-21 cells. The arrow indicates the beginning of IFN treatment. The dashed line indicates the limit of detection.

Fig. 6

Ectopic expression of RIG-I or MDA5 in dKD cells restores their ability to rapidly respond to replication of alphavirus mutants. (A) Comparative levels of RIG-I and MDA5 expression in the parental NIH 3T3 cells and MEFs, treated with IFN-β at a concentration of 500 IU/ml for 24 h, and in selected clones of KI cells. (B) NIH 3T3, dKD, KI RIG-I and KI MDA5 cells were infected with VEEV/GFP/Cm and SINV/G/GFP viruses at an MOI of 20 PFU/cell. Concentrations of released IFN-β were assessed at the indicated times post infection. Means of three biological repeats with SD are presented. (C) The NIH 3T3, KI RIG-I, KI MDA5 and dKD cells were seeded into 6-well Costar plates at a concentration of 5×10⁵ cells per well. SINV/G/GFP and VEEV/GFP/Cm virus stocks were serially diluted and used for infection of indicated cells with different numbers of infectious units. After 1 h incubation at 37°C the virus-containing media were replaced with 2 ml of media supplemented with 0.6% agarose. After incubation at 37°C for 48 h, cells were fixed and plates were scanned on a Typhoon imager to enumerate and assess foci of GFP-expressing cells. Images represent wells infected with the same numbers of infectious units. (D) NIH 3T3, dKD, KI RIG-I and KI MDA5 cells were infected with VEEV/GFP/Cm at an MOI of 20 PFU/cell. Media were replaced every 24 h and cells were split upon reaching confluency in a 1:2 ratio. Virus titers were determined by plaque assay on BHK-21 cells. Dashed line represents the limit of detection. This experiment was repeated twice with similar results.

Fig. 7

Ectopic expression of RIG-I or MDA5 in dKD cells leads to efficient IFN induction in response to replication of VEEV/GFP, which encodes wt capsid protein. (A) NIH 3T3, dKD, KI RIG-I and KI MDA5 cells were infected with VEEV/GFP at an MOI of 20 PFU/cell, and accumulation of IFN-β in the media was assessed at the indicated time points. Means of three biological repeats with SD are presented. (B) NIH 3T3, KI RIG-I, KI MDA5 and dKD cells were infected with VEEV/GFP at an MOI of 20 PFU/cell. Media were replaced at the indicated time points and titers were determined by plaque assay on BHK-21 cells. Means of three biological repeats with SD are presented. (C) NIH 3T3, KI RIG-I, KI MDA5 and dKD cells were seeded into 6-well Costar plates at a concentration of 5×10⁵ cells per well. VEEV/GFP virus stock was serially diluted and used for infection of indicated cells with different numbers of PFUs. After 1 h incubation at 37°C the virus-containing media were replaced with 2 ml of media supplemented with 0.6% agarose. After incubation at 37°C for 48 h, cells were fixed and stained with Crystal Violet. Images represent wells infected with the same numbers of PFUs.

Fig. 8

Ectopic, simultaneous expression of both RIG-I and MDA5 in dKD cells (dKI cell line) differentially affects replication of alpha- and other viruses. (A) Comparative levels of RIG-I and MDA5 expression in MEFs, mock-treated or treated with IFN-β at a concentration of 500 IU/ml for 24 h, and in stable dKI cells. (B) The original dKD and stable dKI cells were seeded into 6-well Costar plates at a concentration of 5×10⁵ cells per well. Stocks of the indicated viruses were serially diluted and used for infection of cells with different numbers of PFUs. After 1 h incubation at 37°C the virus-containing media were replaced with 2 ml of media supplemented with 0.6% agarose. After incubation at 37°C for 48 h, cells were fixed and stained with Crystal Violet. Images represent wells infected with the same numbers of PFUs.