HTML
-
To discover cellular interaction partners of ZIKV E protein, we affinity-isolated proteins from mouse Sertoli cells using a ZIKV EDIII-Fc chimeric protein and we examined these proteins by SDS-PAGE. As shown in Fig. 1, bands of interest in the SDS gel and the overall affinity-isolated proteins eluted from protein A beads were subjected to LC–MS/MS analysis. We thus identified a number of potential interaction partners, including actin and actin-related proteins (Table 1).
Figure 1. Flow chart of identification of ZIKV-EDIII protein interacting proteins. Sertoli cells lysates were incubated with CD80-Fc and protein A-sepharose beads or EDIII-Fc and protein A-sepharose beads, respectively. Samples of CD80-Fc and ZIKV-EDIII-Fc affinity-isolated proteins from Sertoli cell lysates were subjected to gel and performed coomassie blue staining. Band 1 and Band 2 excised from the gel and the whole affinity-isolated proteins eluted from protein A-sepharose beads were all subjected to LC–MS/MS analysis. By performing a sequence alignment with the Uniprot database, we obtained a number of potential interacting proteins, including actin and actin related proteins (Table 1).
Name Mass(kDa) %Cov(95) Accession ZIKV-EDIII-Fc sepharose beads proteins Actin, cytoplasmic 1 41.737 15.73 P60710 Myosin light polypeptide 6 16.930 9.18 Q60605 Band 1 Tubulin beta-5 chain 49.671 22.75 P99024 Beta-actin-like protein 2 42.004 23.94 Q8BFZ3 Band 2 Tropomyosin alpha-1 chain 32.681 70.42 P58771 Actin, cytoplasmic 1 41.737 30.13 P60710 Capping protein (Actin filament) muscle Z-line, alpha 1 32.954 5.59 Q5RKN9 Table 1. LC–MS/MS identified interacting partners of ZIKV-EDIII protein.
-
It has been reported that ZIKV EDIII is the putative region responsible for receptor binding and has an important role in membrane fusion in most flaviviruses (Shi and Gao 2017). In this study, LC-MS/MS identified a number of potential interaction partners of ZIKV EDIII, including actin. The interaction between ZIKV EDIII and actin was verified by Co-IP analysis (Fig. 2A). Meanwhile, Co-IP and reverse Co-IP identified that ZIKV E interacted with actin (Fig. 2B). The amount of E proteins associated actin increased with increasing multiplicity of infection (Fig. 2C), which was consistent with the findings in cells cotransfected with E and actin expression plasmids as shown in Fig. 2B. To determine whether EDIII is the only region participating in the interaction with actin, E truncated constructs were generated (Fig. 2D) and assessed by Co-IP using lysates of transfected Sertoli cells. Surprisingly, ZIKV E regions containing residues 1–133 and 134–301, were both found to bind with actin, suggesting that not only EDIII, but also sequences present in E protein domain Ⅰ (EDⅠ) and/or E protein domain Ⅱ (EDⅡ) interact with actin (Fig. 2E).
Figure 2. ZIKV E interacts with actin. A Co-IP of ZIKV EDIII protein and actin in vitro. Plasmid pcDNA3.1(+)-Flag-EDIII and pcaggs-HA-actin were co-transfected into HEK293T cells, the samples were collected at 48 h post transfection. Cell lysates were immunoprecipitated with anti-mouse immunoglobulin G (IgG) and anti-Flag antibodies, followed by SDS-PAGE and immunoblotting with anti-HA antibody. B Co-IP of ZIKV E protein and actin in vitro. Plasmid pcDNA3.1(+)-Flag-E and pcaggs-HA-actin were co-transfected into HEK293T cells, the samples were collected at 48 h post transfection. Cell lysates were immunoprecipitated with anti-mouse immunoglobulin G (IgG) and anti-Flag antibodies, followed by SDS-PAGE and immunoblotting with anti-HA antibody. HEK293T cells were co-transfected with empty vector, pcDNA3.1(+)-Flag-E and pcaggs-HA-actin, cells lysates were immunoprecipitated with anti-HA antibody followed by SDS-PAGE and immunoblotting with anti-Flag antibody. C Co-IP of ZIKV E protein and actin after viral infection. Sertoli cells were un-infected (mock) or infected with ZIKV at an MOI of 0.1 and 0.5, the samples were collected at 48 hpi. Cell lysate was immunoprecipitated with anti-ZIKV E antibodies followed by SDS-PAGE and immunoblotting with anti-actin antibody. D Diagrammatic representation of ZIKV E protein and three truncated constructs generated in this study. E Co-IP analysis of ZIKV E protein's actin interaction region. HEK293T cells were cotransfected with pcDNA3.1(+)-Flag-E(1–133) and pcaggs-HA-actin or pcDNA3.1(+)-Flag-E(134–301) and pcaggs-HA-actin. Cells lysates were immunoprecipitated with anti-Flag antibodies followed by SDS-PAGE and immunoblotting with anti-HA antibody.
-
Several studies have shown that WNV, JEV, and DENV2 utilize the actin filament network in mammalian cells in the course of infection (Lee and Ng 2004; Henry Sum 2015; Cuartas-Lopez et al. 2018), but direct functional evidence of a role of actin during BTB disruption caused by ZIKV infection is lacking. Thus, Sertoli cells were infected with ZIKV at an MOI of 1, and the actin filament structures were monitored at various time points by confocal microscopy. As shown in Fig. 3A, after 24 h of infection, the organized stress fibers in Sertoli cells were partially disrupted when compared with those in mock control cells, and colocalization of E and F-actin was observed. With the progression of infection, the F-actin configuration in Sertoli cells became disorganized at 48 hpi, actin filaments were no longer distributed evenly across the cytoplasm and most stress fibers were disrupted. These results showed that ZIKV infection disrupts the integrity of the actin filament structure, which might benefit infection.
Figure 3. ZIKV infection induces actin filaments rearrangement in Sertoli cells. A Confocal microscopy analysis of Sertoli cells actin filaments skeleton under continuous ZIKV infection. Sertoli cells were un-infected (mock) or infected with ZIKV (MOI = 1). The cells were fixed in 4% paraformaldehyde at indicated time points, permeabilized, and stained with DAPI to label nuclei (blue), TRITC-phalloidin to label F-actin (red), and ZIKV (green) were detected by FITC-Z6 antibodies. The results are representative of three separate experiments. The scale bar indicates 25 μm. B Confocal microscopy analysis of Sertoli cells actin filaments skeleton after transfected with pcDNA3.1(+)-Flag-E plasmid. Sertoli cells were transfected with 2 μg empty vector, 2 μg pcDNA3.1(+)-Flag-C plasmid or 2 μg empty vector, 2 μg pcDNA3.1(+)-Flag-E plasmid, and fixed at 48 h post transfection. Then cells were stained with DAPI to label nuclei (blue), TRITC-phalloidin to label F-actin (red), Flag antibodies (green) to label capside protein and ZIKV-E (green) were detected by FITC-Z6 antibodies. The results are representative of three separate experiments. The scale bar indicates 25 μm. C Measurements of actin mRNA levels after ZIKV infection. Sertoli cells were un-infected (Mock) or infected with ZIKV at an MOI of 1, 2 and 5, and samples were collected at 30 hpi. Total RNA were extracted, copies of viral RNA and actin were measured by qRT-PCR. The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05; **, P < 0.01; ns, not significant (one-way ANOVA). D Measurements of actin protein levels after ZIKV infection. Sertoli cells were infected with ZIKV (MOI = 1) and collected at indicated time points, actin protein levels were examined by WB. 0 h: un-infected cells.
Next, we investigated whether ZIKV E would be involved in the reorganization of actin filaments caused by ZIKV infection. Confocal immunofluorescence microscopy revealed that transient expression of E proteins in transfected Sertoli cells resulted in the disruption of actin filament structure and the expressed proteins colocalized with actin when compared with empty vector or ZIKV capsid plasmid transfected cells (Fig. 3B). This finding was consistent with the viral infection results, indicating that E protein plays a crucial role in ZIKV infection in addition to host-cell receptor binding and membrane fusion. Besides, ZIKV infection did not alter the mRNA and protein levels of actin, as shown in Fig. 3C, D, indicating that ZIKV infection only induces structural disorganization of F-actin, without altering its expression.
-
To investigate the role of the actin cytoskeleton in ZIKV infection further, Sertoli cells were treated with CytoD, which acts as an actin polymerization inhibitor and disrupts the existing actin cytoskeleton, or Jas, which is a potent inducer of actin polymerization and depletes the cellular pool of free actin monomers available for de novo polymerization (Xiang et al. 2012). A previous study showed that CytoD treatment severely disrupts actin network, increases the number of actin filament ends, and leads to the formation of punctate actin foci in BSC-1 African green monkey kidney cells (Schliwa 1982). We observed similar effects in CytoD-treated Sertoli cells by confocal microscopy, CytoD disrupted the stress fibers and the actin cell cortex as compared to the DMSO control. In Jas-treated Sertoli cells, actin filaments were interrupted and piled up disorderly at the edge of the cell (Fig. 4A), demonstrating the feasibilities of the drugs. CCK8 assays revealed that the Sertoli cell viability was 104%, 100%, 100% and 103% after 48-h incubation with 0.5, 1, 2 and 4 μg/mL CytoD, respectively; and the ratio was 120%, 115%, 120%, and 109% when incubated with 10, 50, 100, and 300 nmol/L Jas, respectively, which were not significantly different from the control (Fig. 4B), indicating that the concentrations of CytoD or Jas used in this study had no effect on cell viability.
Figure 4. ZIKV infection was promoted by CytoD or Jas treatment. A Effects of CytoD and Jas on F-actin. Sertoli cells were treated with 2 μg/mL of CytoD or 200 nmol/L Jas for 26 h. Then cells were stained with DAPI to label nuclei (blue), TRITC-phalloidin to label F-actin (red). Control: DMSO treated cells. The scale bar indicates 25 μm. B Cytotoxicities of CytoD and Jas on Sertoli cells. Sertoli cells were incubated with CytoD or Jas at different concentrations for 48 h, and the cell viability was determined by CCK8 assay. Control: DMSO treated cells. The cell viabilities were expressed as the relative values to control, the results are representative of three separate experiments. Each value represents the mean ± SD of 4 separate replicates. ns, not significant (one-way ANOVA). C Effects of CytoD on ZIKV invasion of Sertoli cells. Sertoli cells were pretreated with DMEM containing 0.4% DMSO (control) or CytoD at 1, 2, 4 μg/mL for 3 h and after that infected with ZIKV (MOI = 1) in the absence of drugs for 2 h, discard the supernatants and wash three times with PBS, the cell samples were collected with TRIzol and measured by qRT-PCR. The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05; ****, P < 0.0001 (one-way ANOVA). D–F Effects of CytoD on ZIKV post entry step. Sertoli cells were incubated with ZIKV (MOI = 1) for 2 h and the supernatants were discarded, washed three times with PBS, then incubated with CytoD at the corresponding concentration. The cell and supernatant samples were collected at 30 hpi. Measured by qRT-PCR, WB and plaque assay. The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant (one-way ANOVA). G Effects of Jas on ZIKV invasion of Sertoli cells. Sertoli cells were pretreated with DMEM containing 0.2% DMSO (control) or Jas at 50, 100, 200 nmol/L for 3 h and after that infected with ZIKV (MOI = 1) in the absence of drugs for 2 h, discard the supernatants and wash three times with PBS, the cell samples were collected with TRIzol and measured by qRT-PCR. The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. **, P < 0.01; ns, not significant (one-way ANOVA). H-J Effects of Jas on ZIKV post entry step. Sertoli cells were incubated with ZIKV (MOI = 1) for 2 h and the supernatants were discarded, washed three times with PBS, then incubated with Jas at the corresponding concentration. The cell and supernatant samples were collected at 30 hpi. Measured by qRT-PCR, WB and plaque assay. The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant (one-way ANOVA).
Sertoli cells were pretreated with CytoD and Jas at different concentrations for 3 h and infected with ZIKV (MOI = 1) for another 2 h in the absence of CytoD or Jas. Then, the cells were washed with PBS three times to remove the uninfected ZIKV and cell samples were collected with TRIzol. As shown in Fig. 4C, G, the amount of invading viruses increased in a dose-dependent manner; viral mRNA levels were 1.3-, 1.22-, and 1.69-fold increased after pretreatment with 1, 2, and 4 μg/mL of CytoD (Fig. 4C), the viral mRNA levels were also increased with increasing Jas concentration (Fig. 4G). These observations were striking, considering that the entry of other flaviviruses into cells was inhibited after CytoD or Jas treatment of the cells (Zhang et al. 2019). Therefore, we further investigated the impact of an interrupted actin cytoskeleton on viral post entry step. Sertoli cells were incubated with ZIKV for 2 h to allow the viruses to fully enter the cells. Then, the viruses were removed by washing and the cells were incubated with CytoD or Jas at the indicated concentrations. Control cells were treated with DMSO. Cell and supernatant samples were collected at 30 hpi. Virus contents in the cells were detected by qRT-PCR and WB, and the supernatants were assessed by qRT-PCR and viral plaque assay. As shown in Fig. 4D, E, the intracellular virus content decreased with increasing drug concentration. In detail, viral mRNA level was decreased by 0.6-fold in cells treated with 2 μg/mL CytoD. Instead, the virus content in the supernatant increased, and this was consistent with the plaque assay results (Fig. 4F). Although there was no difference in intracellular virus content between DMSO and Jas, the extracellular virus content increased significantly after Jas treatment (Fig. 4H–4J). These findings suggested that disturbance of the F-actin network enhanced the production of extracellular virus particles.
The ability of CytoD to enhance ZIKV infection was consistently detected over a range of virus dilutions, ensuring that this enhancement was not a result of the usage of different MOIs (data not shown). Based on the collective findings, we surmised that the disruption of actin filaments dynamics benefits ZIKV infection.
-
According to previous studies, ZIKV has the ability to destroy the BTB, also called the SCB. Meanwhile, researches have implicated that TEER can be treated as a marker for the integrity of the BTB formed by the Sertoli cells (Qiu et al. 2016). Thus, we monitored changes in the TEER during viral infection of an in vitro mSCB model established using highly purified primary mSCs. ZIKV infection (MOI = 5) decreased the TEER slightly at 48 hpi and significantly at 72 hpi (Fig. 5A), which was consistent with our previous research (Hui et al. 2020). Moreover, we have previously confirmed that the decline in TEER was not the result of a decrease in cell viability caused by viral infection (Hui et al. 2020). Meanwhile, it has been reported that CytoD disrupts actin filaments and TJ barriers of intestinal epithelial cells (Fu et al. 2008), and that TNF-α mediated restructuring of the SCB in vitro involves actin cytoskeleton reorganization (Lydka et al. 2012). We observed a similar phenomenon in the in vitro mSCB model, CytoD or TNF-α treatment perturbed the SCB in a time- and dose-dependent manner when compared with the control treatment (Fig. 5B). In addition, a reduction in TEER was detected at 48 h post transfection in cells overexpressing a higher amount of protein E (Fig. 5C), indicating a direct effect of E protein on the BTB. These results demonstrated that both viral infection and ectopic expression of E protein could compromise the integrity of an mSCB model in a time- and dose-dependent manner, which may account for the testicular destruction observed in ZIKV-infected mice.
Figure 5. ZIKV E participates in the hyperpermeability of the in vitro mSCB model. A Effects of ZIKV infection on in vitro mSCB model. Primary mSCs were cultured on Transwell semipermeable membranes (0.4 μm pore size). Un-treated (mock) or treated with ZIKV at different MOIs when TEER values exceeds 50 Ω • cm2 and remains unchanged, detected TEER values at indicated time. Relative TEER: (Ωexperimental condition − Ωmedium alone)/(Ωmock − Ωmedium alone). The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05; **, P < 0.01 (two-way ANOVA). B Effects of the CytoD and TNF-α on in vitro mSCB model. CytoD (Control: DMEM containing 0.1% DMSO-treated) and TNF-α (Control: DMEM-treated) were treated on in vitro mSCB model and detected TEER values at indicated time. Relative TEER: (Ωexperimental condition − Ωmedium alone)/(Ωmock − Ωmedium alone). Each value represents the mean ± SD of 3 separate replicates. P values were analysed by comparing with the corresponding controls of 1 μg/mL CytoD and 1 ng/mL TNF-α respectively. ****, P < 0.0001 (two-way ANOVA). C Effects of ZIKV E overexprssion on in vitro mSCB model. Empty vector (mock) or pcDNA3.1(+)-Flag-E plasmid at different concentrations was transfected into mSCs, and TEER values were detected at different time points as indicated. Relative TEER: (Ωexperimental condition − Ωmedium alone)/(Ωmock − Ωmedium alone). The results are representative of three separate experiments. Each value represents the mean ± SD of 3 separate replicates. *, P < 0.05 (two-way ANOVA). D Co-IP of actin and ZO-1 after ZIKV infection and ZIKV E overexprssion. Sertoli cells were cultured overnight in 10 cm dishes, then un-treated (mock) or treated with ZIKV and transfected with empty vector or pcDNA3.1(+)-Flag-E plasmid at different concentrations, samples were collected after 48 h treatment. Cell lysate was immunoprecipitated with anti-ZO-1 or anti-mouse immunoglobulin G (IgG) antibody followed by SDS–PAGE and immunoblotting with anti-actin antibody. E Confocal microscopy analysis of the localization of ZO-1 after ZIKV infection and ZIKV E overexpression. Sertoli cells were un-treated (mock) or treated with ZIKV (MOI = 1) and transfected with 2 μg empty vector or 2 μg pcDNA3.1(+)-Flag-E plasmid. Then cells were fixed after 48 h treatment and stained with DAPI to label nuclei (blue), TRITC-phalloidin to label F-actin (red), and ZO-1 (green) were detected by anti-ZO-1 antibodies. The results are representative of three separate experiments. The scale bar indicates 25 μm. F Identification of E transfection efficiency in primary mSCs. Primary mSCs were transfected with empty vector or pcDNA3.1(+)-Flag-E plasmid and collected at 48 h after transfection, the expression of E, actin and ZO-1 was measured by WB.
SCB function is supported by the binding of the organized actin cytoskeleton to adaptor proteins (Mok et al. 2013). As mentioned above, ZIKV infection and ectopically expressed E proteins induced the reorganization of F-actin network and decreased the TEER in an in vitro mSCB model. Thus, we hypothesized the disruption of the SCB was mediated by reorganization of F-actin. To validate this hypothesis, Co-IP and immunofluorescence assays were conducted. As shown in Fig. 5D, the association of actin with the TJ adaptor protein ZO-1 decreased in a dose-dependent manner in Sertoli cells after ZIKV infection or ZIKV E transfection, prompting that the weakened interaction between actin and ZO-1 might have contributed to the decrease in TEER. Furthermore, the continuous signal of ZO-1 at the cell–cell border was interrupted in infected or transfected Sertoli cells, as part of the proteins were translocated from the cell surface to the cytoplasm, when compared with control cells (Fig. 5E). As shown in Fig. 5F, overexpression of E in primary Sertoli cells did not affect the protein levels of actin and ZO-1. However, it is worth noticing that since F-actin was depolymerized and ZO-1 was diffused from the plasma membrane to the cytoplasm, the colocalization of the two proteins seems more obvious in treated cells. Nonetheless, these results suggested that the destruction of the organized actin cytoskeleton caused by ZIKV E contributed to the decline in TEER values, supporting the notion that ZIKV infection disrupts the SCB through the reorganization of F-actin, and identifying E protein as an important viral molecule in this process.