Matrix metalloproteinase 3 promotes cellular anti-dengue virus response via interaction with transcription factor NFκB in cell nucleus

Abstract

Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.

Figure 1. Mmp3 is upregulated upon DENV infection.

A. Mouse Mmp3 expression in DENV-2 infected or uninfected RAW264.7 and MEF cells were analyzed by quantitative RT-PCR (qRT-PCR), and normalized to mouse beta actin gene. Results are expressed as the mean + the SEM. * p<0.05 and ** p<0.01 (t-test). B. The protein level of mouse MMP3 was increased in DENV infected RAW264.7 cells (MOI = 1.0, 48 hrs post infection) compared with uninfected cells. C. Mouse Mmp3 mRNA expression in DENV-2 infected or uninfected mouse peritoneal macrophages. (MOI = 1.0, 48 hrs post infection). D. Human MMP3 mRNA expression increased in human PBMC, 293T and A549 cells upon DENV infection. (MOI = 1.0, 48 hrs post infection, respectively). Representative results from at least 3 independent experiments.

Figure 2. The antiviral effect of Mmp3 against DENV.

A-B) RNAi efficiency for Mmp3 as shown in qRT-PCR (A) and Western blot (B). C-D) DENV burdens in RAW264.7 cells after RNAi silencing. C) The viral burdens were analyzed by measuring the virus E gene copy using qRT-PCR, and normalized to mouse beta actin gene. D) The titer of infectious DENV in cell supernatants 24 hrs post infection, as measured by TCID₅₀ assay. E) The viral burdens in Mmp3 overexpressed cells were decreased comparing with that in control cells. Results are expressed as the mean + the SEM. * p<0.05 and ** p<0.01(t-test). Representative results from at least 3 independent experiments.

Figure 3. Cytokine expression in DENV infected RAW264.7 cells after gene silencing.

A–F) mRNA levels of selective cytokines/chemokines (A: Ifnβ1, B: Il 6, C:Cxcl1,D:Cxcl2,E:Ccl5,F:Ccr5) were measured by qRT-PCR and normalized to mouse beta-actin gene. G–H) Protein level of TNFα and IL6 in cell supernatants. Results are expressed as the mean + the SEM. * p<0.05 and ** p<0.01. (t-test) Representative results from at least 3 independent experiments.

Figure 4. MMP3 moves from cell cytoplasm into nucleus upon DENV infection and influence NFκB activity.

A) MMP3 moves from cell cytoplasm into nucleus upon DENV infection. B) The cytoplasmic and nuclear distribution of overexpressed flag-MMP3 in DENV infected or uninfected cells as determined by Western Blot. C)NFκB transcriptional activity is impaired upon MMP3 silencing in DENV infected 293T,A549 and RAW264.7 cells, while AP1 transcriptional activities were not influenced. D and E) Overexpression of MMP3 activates NFκB activity in DENV infected RAW264.7 (D) and 293T(E) cells. MMP3 activated NFκB in a dose dependent manner in DENV infected 293T cells(E) (MOI = 1.0, 24 hrs post infection). (The reporter activities were normalized by internal control (pRL-TK Renilla luciferase value). The mean value of activities from DENV infected control cells were set to 1.0) Results are expressed as the mean + the SEM. * p<0.05 and ** p<0.01 (t-test). Representative results from at least 3 independent experiments.

Figure 5. MMP3 interacts with NFκB in cell nucleus upon DENV infection.

A) Co-localization of MMP3 and NFκB RelA(p65) in cell nucleus upon DENV infection. B and C) NFκB RelA (p65) and P50 were co-immunoprecipitated with MMP3 in total cell extract from DENV infected cells. The co-IP was carried out using anti-flag affinity gel and the bound proteins were detected by immunoblotting with anti-GFP, anti-P50 and anti-flag antibodies, respectively. D) NFκB RelA (p65) and P50 were co-immunoprecipitated with MMP3 in cell nucleus upon DENV infection. Representative results from at least 3 independent experiments.

Figure 6. The C-terminal hinge and hemopexin-like domain of MMP3 is required for interaction with NFκB.

A) Domain structure of MMP3 and the truncated MMP3 used in this study. B) Co-immunoprecipitation assay with various domains of MMP3 and NFκB (RelA(p65) and P50). C) Influence of NFκB activity by various domains of MMP3 measured by Luciferase reporter assays. Results are expressed as the mean + the SEM. * p<0.05, when compared with controls(t-test). Representative results from at least 3 independent experiments.