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. 2018 Nov 21;7:e39494. doi: 10.7554/eLife.39494

Figure 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).

Figure 2.

Figure 2—figure supplement 1. Active RV replication induces proteasomal degradation of MAVS.

Figure 2—figure supplement 1.

(A) MA104 cells were infected with RRV (MOI = 1) and harvested at indicated time points post infection for RT-qPCR analysis. MAVS mRNA levels were measured and normalized to that of GAPDH. (B) African green monkey COS-7 cells were infected with RRV (MOI = 3) and harvested at indicated time points for western blot (top panel). MA104 cells were infected with different RV strains (MOI = 3) and harvested at 8 hpi for western blot (middle panel). HT-29 cells were infected with Wa at MOI = 1 or 10 for indicated time points and harvested for western blot. (RRV, SA11: simian RVs; UK: bovine RV; OSU, SB1A: porcine RVs; Wa, DS1: human RVs; ETD: murine RV). (C) MA104 cells were infected with RRV (MOI = 3), with RRV mixed with neutralizing antibodies (nAb) or with psoralen UV-inactivated RRV (UV) for 6 or 12 hpi. The lysates were harvested and examined by western blot. (D) Supernatants were harvested from mock-infected MA104 cells (sup(M)) or RRV-infected MA104 cells (sup(R)) and used to re-infect fresh MA104 cells. Lysates were harvested at 6 or 12 hpi and examined by western blot. (E) MA104 cells were infected with RRV (MOI = 3) for 12 hpi in the presence of different chemical inhibitors. (ITZ: itraconazole; Salu: salubrinal; EerI: Eeyarestatin I; Spi: spiroepoxide; GW: GW4869; STS: staurosporine; Z-VAD: Z-VAD-FMK). (F) MA104 cells were infected with RRV (MOI = 3) and treated with MG132 at indicated time point post infection (top panel). MA104 cells were infected with RRV (MOI = 3) for 12 hpi in the presence of different chemical inhibitors. (MG: MG132; lact: lactacystin; chlo: chloroquine; conA: concanamycin A; Z-VAD: Z-VAD-FMK; 3-MA: 3-Methyladenine; rap: rapamycin). 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).