Over-expression of mitochondrial antiviral signaling protein inhibits coxsackievirus B3 infection by enhancing type-I interferons production

Abstract

Background: Recent studies have revealed that Mitochondrial Antiviral Signaling (MAVS) protein plays an essential role in the inhibition of viral infection through type I interferon (IFN) pathway. It has been shown that 3C (pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism.

Results: In this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3) were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE) and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs.

Conclusions: In summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

Figure 1

Time and dose effect of MAVS on cell growth and IFN-β production. HeLa cells were transfected with pcDNA 3.1 and pcDNA3.1-FLAG-MAVS. (A) At 24 h post-transfection, fusion protein of FLAG-MAVS was detected by western blotting. (B) Cell proliferation was evaluated through trypan blue dye exclusion. (C) At 24 h post-transfection, RT-qPCR was performed to evaluate the IFN-β transcriptional level at different dosages of MAVS construct transfection. (D) The concentration of IFN-β in culture supernatant was determined with ELISA at different time points post-transfection. * p < 0.05,** p < 0.01.

Figure 2

Restriction of CVB3 caused CPE by exogenous MAVS in HeLa cells. At 24 h post-transfected with p-EGFP or p-MAVS-EGFP, HeLa cells were challenged by CVB3-mCherry. After 24 h and 48 h of CVB3-mCherry infection, (A) the expression of MAVS-GFP (green), CVB3-mCherry infection (red) and the CPE were observed. (B) The percentage of infected cells was calculated. (C) CVB3-mCherry was used to inoculate HeLa cells (MOI = 0, 0.001, 0.01, 0.1, 1) after transfection with p-EGFP or p-MAVS-EGFP. MTT test was used to determine the cell viability. (D &E) Cells over-expressing exogenous MAVS were exposed to CVB3-woodruff or CVB3-mCherry respectively, and the survival cells were stained by crystal violet. The percentage of survival cells was evaluated by observation. (F) HeLa cells were transfected with p-EGFP or p-MAVS-EGFP for 24 h and harvested for RNA extraction, and RT-qPCR was performed to verify the expression level of exogenous MAVS. * p < 0.05,** p < 0.01.

Figure 3

Inhibition of CVB3 by over-expressing MAVS, coinciding with IFNs increase. After transfection with pcDNA3.1 and pcDNA3.1-FLAG-MAVS, HeLa cells were infected with CVB3-woodruff. At 0 h, 6 h, 12 h, 24 h and 36 h post-infection, cells were harvested, and RT-qPCR was performed to determine the change of (A) CVB3 replication, (B) IFN-β mRNA and (C) IFN-α mRNA. * p < 0.05,** p < 0.01.

Figure 4

Inhibition of CVB3 by exogenous MAVS in pre-infected HeLa cells. HeLa cells were challenged by CVB3-mCherry (absorption for 1 h) and then transfected with p-EGFP and p-MAVS-EGFP. (A) MAVS-GFP expression (green), CVB3 infection (red) and CPE were observed at 24 h and 48 h. (B) The percentage of infected cells with red fluorescence was calculated. (C) MTT was performed to determine the cell viability of HeLa cells inoculated with CVB3 at MOI of 0, 0.001, 0.01, 0.1, 1 and then transfected with p-EGFP and p-MAVS-EGFP plasmids (48 h post-transfection). * p < 0.05,** p < 0.01.

Figure 5

Inhibition of CVB3 and up-regulation of IFNs by MAVS introduction after CVB3 infection. After exposure to CVB3 for 1 h, pcDNA3.1 and pcDNA3.1-FLAG-MAVS were introduced into HeLa cells. In 0 h ~ 36 h, cells were harvested and total RNA was extracted. (A) CVB3 RNA, (B) IFN-β mRNA and (C) IFN-α mRNA were detected through RT-qPCR. * p < 0.05,** p < 0.01.