Matrix metalloproteinase 9 facilitates Zika virus invasion of the testis by modulating the integrity of the blood-testis barrier

Abstract

Zika virus (ZIKV) is a unique flavivirus with high tropism to the testes. ZIKV can persist in human semen for months and can cause testicular damage in male mice. However, the mechanisms through which ZIKV enters the testes remain unclear. In this study, we revealed that matrix metalloproteinase 9 (MMP9) was upregulated by ZIKV infection in cell culture and in A129 mice. Furthermore, using an in vitro Sertoli cell barrier model and MMP9-/- mice, we found that ZIKV infection directly affected the permeability of the blood-testis barrier (BTB), and knockout or inhibition of MMP9 reduced the effects of ZIKV on the Sertoli cell BTB, highlighting its role in ZIKV-induced disruption of the BTB. Interestingly, the protein levels of MMP9 were elevated by ZIKV nonstructural protein 1 (NS1) in primary mouse Sertoli cells (mSCs) and other cell lines. Moreover, the interaction between NS1 and MMP9 induced the K63-linked polyubiquitination of MMP9, which enhanced the stability of MMP9. The upregulated MMP9 level led to the degradation of essential proteins involved in the maintenance of the BTB, such as tight junction proteins (TJPs) and type Ⅳ collagens. Collectively, we concluded that ZIKV infection promoted the expression of MMP9 which was further stabilized by NS1 induced K63-linked polyubiquitination to affect the TJPs/ type Ⅳ collagen network, thereby disrupting the BTB and facilitating ZIKV entry into the testes.

Fig 1. MMP9 was upregulated by ZIKV in vivo.

A129 male mice (8 weeks old, n = 5) were infected with ZIKV (1×10⁶ PFU) for 0, 2, 4, 6, 8, or 10 days. (A and C) MMP9 mRNA expression levels (A) and MMP2 mRNA expression levels (C) in the whole blood were measured by qRT-PCR and normalized to the GAPDH mRNA level. (B) A probe-based assay was used to quantify viral RNA copy number by TaqMan qPCR amplification of ZIKV E gene. (D and F) MMP9 mRNA expression levels (D) and MMP2 mRNA expression levels (F) in the testes were examined by qRT-PCR. (E) A probe-based assay was used to quantify viral RNA copy number by TaqMan qPCR amplification of ZIKV E gene. (G) Protein levels of MMP9 in the sera were tested by ELISA. Data are expressed as means ± SEMS of three independent experiments. *P< 0.05; **P< 0.01; ***P< 0.001; ****P<0.0001. ns, not significant (one-way ANOVA). (H) Protein levels of MMP9, MMP2, NS1, GAPDH in the testes were quantified by western blotting (top), and proteinase activity was examined by gelatin zymography (bottom). Transparent bands indicate the presence of active MMP9 or MMP2. (I) Results of immunofluorescence staining. A129 mouse testis tissues were stained with DAPI to label nuclei (blue), an antibody against MMP9 (red), and ZIKV-positive signals (green) were detected by Z6 antibodies. Representatives of the tubules within the seminiferous epithelium are indicated with white dotted lines. Scale bar, 50μm.

Fig 2. MMP9 compromised BTB integrity to facilitate ZIKV entry into the testes.

C57BL/6 WT and MMP9^-/- male mice (6–7 weeks old) treated with Ifnar-blocking mouse monoclonal antibodies were infected intraperitoneally with ZIKV (1 × 10⁷ PFU). The C57BL/6 WT mice treated with isotype control antibodies were also infected intraperitoneally with ZIKV (1 × 10⁷ PFU) as a mock control. (A) RNA was extracted from the whole blood, and a probe-based assay was used to quantify viral RNA copy number by TaqMan qPCR amplification of ZIKV E gene at different time points. Data shown are means ± SEMs; ns, not significant, n = 5. (two-tailed Student’s t-tests). (B) RNA was extracted from the testes, and a probe-based assay was used to quantify viral RNA copy number by TaqMan qPCR amplification of ZIKV E gene at different time points. Data shown are means ± SEMs; *P< 0.05; **P< 0.01; ***P< 0.001, n = 5. (one-way ANOVA). (C and D) Results of immunofluorescence staining for ZIKV (green) and CD45 (red) in the testes and the quantifications were shown using Image J. Data shown are means ± SEMs; *P< 0.05; **P< 0.01; ***P< 0.001, ****P< 0.0001, n = 5 (one-way ANOVA), Scale bar, 50μm. (E) Histopathological changes in the testes of WT and MMP9^-/- mice on day 10 post infection. Disrupted seminiferous tubules with leukocyte infiltration (blue arrow), abnormally organized cells (red arrow), and intratesticular congestion (black arrow) were obviously observed in ZIKV-infected WT testes. Representative images from several independent experiments are shown, Scale bar, 100μm. (F and G) Results of immunofluorescence staining of occludin (red), ZO-1 (red), claudin-1 (red), and type Ⅳ collagen (red) and the quantifications for the percentage of these proteins were shown using Image J. Data shown are means ± SEMs; *P< 0.05; **P< 0.01, n = 5 (one-way ANOVA), Scale bar, 50μm. (H) Evans blue BTB permeability. On day 10 postinfection, WT and MMP9^-/- mice were injected with Evans blue and perfused 1h later. Uninfected WT mice were used as a control. Data are representatives of the results of three experiments (n = 8/group). (I) Quantification of Evans blue in the mouse testes. Evans blue was extracted from whole testes, and absorbance was measured, using uninfected-mouse testis extracts as a blank. *P< 0.05; **P< 0.01. ns, not significant (one-way ANOVA).

Fig 3. ZIKV infection induced the expression and activity of MMP9 and altered barrier integrity.

(A–C) Primary mSCs (A), primary MEFs (B), and A549 cells (C) were infected with ZIKV at a MOI of 1. MMP9 mRNA levels were measured by quantitative RT-PCR (top), MMP9 protein levels were examined by western blotting (middle), and MMP9 proteinase activity was determined by gelatin zymography assays (bottom). Data are expressed as means ± SEMs of three separate experiments. *P< 0.05; **P< 0.01; ***P< 0.001. ns, not significant (one-way ANOVA). (D) Primary mSCs were cultured on Transwell semipermeable membranes (0.4 μm pore size). At day 3, Sertoli cells with an established functional tight junction barrier were infected with ZIKV (MOI = 5) and then treated with or without a specific inhibitor of MMP9 (JNJ0966; 1μM). The untreated cells were used as a mock control. The integrity of the SCB model was determined by measuring TEER at each time point. TEER values were expressed in Ohms (Ω). (E) Primary mSCs were cultured on Transwell semipermeable membranes (0.4 μm pore size), activated MMP9 (50ng/mL) were added and then treated with or without JNJ0966 (1μM). The integrity of the SCB model was determined by measuring TEER at each time point. The data are expressed as means ± SEMs of three independent experiments. *P< 0.05; **P< 0.01; ***P< 0.001. ns, not significant (one-way ANOVA).

Fig 4. MMP9 was upregulated by ZIKV NS1 on protein level.

(A–J) HEK293T cells were transfected with pcDNA3.1(+)-Flag-C, -M, -E, -NS1, -NS2A, -NS2B, -NS3, -NS4A, -NS4B, or -NS5 using a concentration gradient for 48h. Total MMP9, C, M, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5, and GAPDH levels in whole cell lysates (WCLs) were determined by western blotting with the indicated antibodies. MEFs (K) and A549 cells (L) were transfected with different concentrations of pcDNA3.1(+)-Flag-NS1. After 48h post tranfection total MMP9, NS1, and GAPDH proteins expressed in WCLs were detected by western blotting with the indicated antibodies. (M–O) Different concentrations of pcDNA3.1(+)-Flag-NS1 were transfected into primary mSCs (M), A549 cells (N), and HEK293T cells (O). After 48h post tranfection, MMP9 mRNA levels were measured by qRT-PCR. Data are expressed as means ± SEMs of three independent experiments. ns, not significant (one-way ANOVA). (P) Primary mSCs were transfected with empty vector or pcDNA3.1(+)-Flag-NS1, and resistance values were examined from day 2 after transfection. Data are expressed as means ± SEMs of three independent experiments. *P< 0.05. ns, not significant (two-way ANOVA). (Q) Primary mSCs were transfected with empty vector or pcDNA3.1(+)-Flag-NS1, and the expression of NS1 and MMP9 was measured by western blotting.

Fig 5. NS1 bound to MMP9 and facilitated K63-linked polyubiquitination of MMP9.

(A) HEK293T cells were cotransfected with empty vector, plasmid pcDNA3.1(+)-Flag-NS1 expressing Flag-tagged NS1 and plasmid pCAGGS-HA-MMP9 expressing HA-tagged MMP9. 48h later, cells were treated with 40μg/mL CHX for 0, 9h, and then cells were lysed, and subjected to western blot with indicated antibodies. (B) HEK293T cells were cotransfected with empty vector/ Flag-NS1 and HA-MMP9 for 30h. Cells were treated with 40μg/mL CHX for 0, 9h, then immunostained with anti-Flag (red) and anti-HA (green) antibodies, and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy. Scale bar, 10μm. (C-D) HEK293T cells were cotransfected with empty vector, Flag-NS1 and HA-MMP9, cells lysates were immunoprecipitated with anti-HA (C) or anti-Flag (D) antibodies. The immunoprecipitates and whole-cell lysates (WCLs) were analyzed by western blotting with anti-Flag and anti-HA antibodies. (E) HEK293T cells were cotransfected with plasmid Flag-NS1 and HA-MMP9. Cells were lysed, and cell lysates were immunoprecipitated with anti-Flag and anti-mouse immunoglobulin G (IgG) antibodies. The immunoprecipitates and whole-cell lysates (WCLs) were analyzed by western blotting with anti-Flag and anti-HA antibodies. (F) Primary MEFs were infected with ZIKV at an MOI of 1 for 30h, and cell lysates were immunoprecipitated with anti-MMP9 and anti-goat IgG antibodies. The immunoprecipitates and WCLs were analyzed by western blotting with anti-MMP9 and anti-NS1 antibodies. HeLa (G) and HEK293T cells (H) were cotransfected with Flag-NS1 and HA-MMP9 for 24h. Cells were immunostained with anti-Flag (red) and anti-HA (green) antibodies, and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy. Scale bar, 10μm. (I) Immunoblot analysis of extracts of HEK293T cells transfected with plasmid for HA-MMP9 and Flag-NS1 and treated with dimethylsulfoxide (DMSO) or MG132. (J) HEK293T cells were cotransfected with pGFP-MMP9, pHA-Ub, and pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-HA antibodies. (K) HEK293T cells were cotransfected with pGFP-MMP9, pHA-K63, and pHA-K48, together with pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with anti-HA antibodies. (L) HEK293T cells were cotransfected with pGFP-MMP9 and pHA-K63R or pHA-K48R, together with pFlag-NS1. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotted with anti-HA antibodies.

Fig 6. The possible mechanisms through which ZIKV enters the testes.

Under normal conditions, earlier germ cells are continually separated from the blood supply into the lumen of the seminiferous tubules. The BTB, which localizes basally between adjacent SCs, is closest to the basement membrane, i.e., a type of pericellular matrix that is stabilized by a collagen type Ⅳ network. The testes are in an immunoprivileged environment. During ZIKV infection, Leydig cells, SCs, and immune cells (e.g., macrophages, T cells, and dendritic cells) are infected with ZIKV, changing the microenvironment from an immunosuppressive environment to a strong antiviral environment. Activated macrophages secrete cytokines, such as tumor necrosis factor α, which can also disrupt the BTB by degradation of TJPs, allowing ZIKV to reach and infect germ cells. During the infection course, large amounts of MMP9 in the sera or testes are induced and translocate to the basement membrane, where it degrades its target proteins, including TJPs and type Ⅳ collagen, thereby destroying the integrity of the BTB. NS2A may also interact directly with adherent junction components, targeting them for lysosomal degradation as it does in mouse radial glial cells and human brain organoids. In addition, ZIKV may be carried in the infected macrophages through the BTB into the lumen of the seminiferous tubules. Intracellular NS1 can enhance the stability of MMP9 by promoting K63-linked polyubiquitination of MMP9.