Fig 2: RV infection induces MAVS degradation in a host range restricted manner.(A) WT and MAVS-/- HT-29 cells were infected with the simian RV RRV strain or the human RV Wa strain (MOI = 1) for 24 hr. Expression of viral gene NSP5 was measured by RT-qPCR and normalized to that of GAPDH. The virus yield in the supernatant was harvested and measured by a standard focus forming unit assay. (B) MA104 cells were infected with RRV at indicated MOIs for 6 or 12 hr. The lysates were harvested for western blot to examine the expression level of full-length (FL) and truncated mini-MAVS, other indicated RIG-I signaling factors, RV proteins (VP4, VP6) and GAPDH. The relative levels of individual proteins were quantified (quant) with respect to the GAPDH levels (lane 1 set as 1.0). (C) Rhesus MA104 cells, human HT-29 cells and murine NIH3T3 cells were infected with simian RRV, human Wa or murine ETD (MOI = 5) respectively and the protein levels of MAVS, VP6 and IRF3 were measured by western blot (UI: uninfected). (D) HEK293 cells were transfected with Flag-tagged MAVS from Homo sapiens (human, hu), Chlorocebus aethiops (African Green monkey, AGM), Macaca mulatta (rhesus monkey, rh) for 12 hr and infected with Wa (MOI = 3) for 12 hr. The protein levels of MAVS, VP6 and GAPDH were measured by western blot. (E) MA104 cells were transfected with luciferase expression constructs driven by IFN-β, NF-κB, or ISRE promoters, mock or infected with RRV (MOI = 3) for 8 hr, and stimulated with LMW poly (I:C) (pIC) for 8 hr. The level of firefly luciferase (FFL) was measured and normalized to that of renilla luciferase (RL), which serves as internal control. (F) MA104 cells were infected with RRV (MOI = 3) for 12 hr and treated with indicated proteasome and lysosome inhibitors for 12 hr. The lysates were harvested and MAVS level was measured by western blot. (MG: MG132; epo: epoxomicin; lac: lactacystin; conA: concanamycin A; bort: bortezomib). For all figures, experiments were repeated at least three times with similar results. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001). (* represents mini-MAVS in all western blots).

Fig 3: Phosphorylation mediates MAVS degradation during RV infection.(A) Schematic diagram of MAVS and chimeric EGFP-MAVS proteins with defined domains illustrated in colors. Two lysines within the CARD domain and novel post-translational modifications (PTMs) are pointed out (left panel). (B) MA104 cells were transfected with indicated MAVS plasmids for 48 hr with or without RRV infection (MOI = 1) for the last 12 hr. The lysates were harvested and examined by western blot. (C) MA104 cells were co-transfected with GFP-tagged MAVS domain mutants and V5-tagged RRV VP3 for 48 hr and lysates were harvested for immunoprecipitation using anti-V5 antibody. (D) MA104 cells were transfected with indicated GFP-tagged MAVS mutants for 48 hr with or without RRV infection (MOI = 1) for the last 12 hr. The levels of MAVS and viral protein VP6 expression were measured by western blot. (E) MA104 cells were transfected with WT or indicated lysine-mutants of HA-tagged ubiquitin for 48 hr, infected with RRV (MOI = 1) for the last 12 hr and harvested for immunoprecipitation using an anti-MAVS antibody. The levels of ubiquitin-conjugated MAVS were measured using an anti-HA antibody. (F) Mass spectrometry analysis of PTMs on MAVS, immunoprecipitated from RRV-infected MA104 lysates at six hpi. Numbers indicate the mass increase (+79.97: phosphorylation;+14.02: methylation). The novel PTMs identified in this study are highlighted in red. The percentage of phosphorylation and methylation modification in total MAVS proteins is presented as %. (G) MA104 cells were transfected with WT or MAVS mutants (SPLTSS: SPLTSS motif mutated to six alanines; R3A: R232, R236, R239 mutated to three alanines) for 48 hr with or without RRV infection (MOI = 1) for the last 12 hr. The levels of MAVS were measured by western blot. (H) HEK293 cells were co-transfected with GFP-tagged Wa-VP3 and WT or MAVS mutants for 48 hr and treated with MG132. Lysates were harvested for immunoprecipitation using anti-GFP antibody and probed for MAVS levels. For all figures except (e) and (f), experiments were repeated at least three times with similar results. Experiment in (e) was performed twice and (f) was performed once.

Fig 4: Replication of heterologous RV is enhanced in selected knockout mouse strains.(A) Viral antigens in the fecal samples of EW or RRV-infected WT and Mavs-/- mice were measured by a standard ELISA assay over the course of 12 days. (B) Small intestines, liver and mesenteric lymph node (MLN) were harvested at 3 dpi or 7 dpi from EW or RRV infected animals and the levels of viral NSP5, indicative of virus replication, was measured by RT-qPCR and normalized to that of GAPDH. (C) C57Bl6/J mice with indicated gene deficiencies were infected with RRV (107 pfu) or EW (104 DD50). Three days later, feces samples were collected and tested by an ELISA assay for viral antigens and the amount of viruses were converted to PFU equivalents based on the standard curve. At least 8 mice were used in each group. For all figures, experiments were repeated at least two times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).10.7554/eLife.39494.013Figure 5—source data 1.Source data for Figure 5A.10.7554/eLife.39494.014Figure 5—source data 2.Source data for Figure 5B.10.7554/eLife.39494.015Figure 5—source data 3.Source data for Figure 5C.

Fig 5: VP3 mediates MAVS interaction and degradation using an N-terminal domain.(A) MA104 cells were transfected with plasmids expressing each of the 12 GFP-tagged RRV proteins for 72 hr and then subjected to sorting for GFP positive and negative cells. The levels of endogenous full-length and mini-MAVS were normalized to those of GFP-conjugated RRV proteins. (B) MA104 cells were co-transfected with 0.5 μg of Flag-tagged rhesus MAVS and GFP-tagged RRV-VP3 or RRV-NSP1 plasmids for 48 hr. RRV infection (MOI = 1) for 12 hr serves as a positive control. Total cell lysates were harvested and examined by western blot by indicated antibodies. (C) MA104 cells were infected with RRV (MOI = 3) for 8 hr in vehicle control (DMSO) or MG132 (10 μM) treatment. The lysates were subjected to immunoprecipitation using anti-MAVS antibody and analyzed by mass spectrometry for viral proteins. (D) MA104 cells were co-transfected with Flag-tagged MAVS and GFP-tagged RRV-VP3 or RRV-NSP4 for 48 hr and lysates were precipitated with anti-Flag antibody and GFP levels were examined by western blot. (E) Schematic diagram of WT and mutant VP3 proteins with defined domains illustrated in colors and catalytic sites of phosphodiesterase (PDE) activity indicated (left panel). MA104 cells were co-transfected with Flag-tagged MAVS and GFP-tagged VP3 mutants for 48 hr. The lysates were harvested and the levels of Flag and GFP were measured by western blot. (F) MA104 cells were co-transfected with Flag-tagged MAVS and GFP-tagged RV proteins (WT VP3, N-terminal K172* VP3, and NSP4) for 48 hr and lysates were harvested for immunoprecipitation using anti-GFP antibody. (G) MA104 cells were transfected with GFP-tagged wild-type or PDE mutant RRV-VP3 for 72 hr and then subjected to sorting for GFP positive cells. The levels of endogenous MAVS and GFP were examined by western blot. (H) MA104 cells were co-transfected with Flag-tagged MAVS and GFP-tagged VP3 from RRV (simian), Wa (human), ETD (murine) RV strains or chimeric RRV VP3 with 171 amino acids from ETD VP3, treated with MG132. The lysates were harvested for immunoprecipitation using anti-Flag antibody and probed for GFP levels. (I) MA104 cells were co-transfected with Flag-tagged MAVS and GFP-tagged RRV-VP3, and treated with MG132 (MG) or chloroquine (chlo) for 12 hr. The levels of Flag and IRF3 were examined by western blot. For all figures except (c), experiments were repeated at least three times with similar results. Experiments in (c) were performed once. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*p≤0.05; **p≤0.01; ***p≤0.001).10.7554/eLife.39494.008Figure 3—source data 1.Source data for Figure 3C: MAVS-RV protein IP counts.