Erp57 facilitates ZIKV-induced DNA damage via NS2B/NS3 complex formation

Abstract

It is believed that DNA double-strand breaks induced by Zika virus (ZIKV) infection in pregnant women is a main reason of brain damage (e.g. microcephaly, severe brain malformation, and neuropathy) in newborn babies [1,2], but its underlying mechanism is poorly understood. In this study, we report that the depletion of ERp57, a member of the protein disulphide isomerase (PDI) family, leads to the limited production of ZIKV in nerve cells. ERp57 knockout not only suppresses viral induced reactive oxygen species (ROS) mediated host DNA damage, but also decreases apoptosis. Strikingly, DNA damage depends on ERp57-bridged complex formation of viral protein NS2B/NS3. LOC14, an ERp57 inhibitor, restricts ZIKV infection and virus-induced DNA damage. Our work reveals an important role of ERp57 in both ZIKV propagation and virus-induced DNA damage, suggesting a potential target against ZIKV infection.

Keywords: DNA damage; ERp57; ROS; ZIKA virus; ZIKV NS2B/NS3 complex; antiviral; apoptosis.

Figure 1.

ERp57 depletion suppresses ZIKV production in vitro. (A) Wild type RD and ERp57-KO cells were infected with ZIKV (MOI = 1). Intracellular RNA samples were collected at the indicated time post infection, later used for quantitative RT-PCR. (B) Representative CPE images of wild type or ERp57-KO RD cells at 72 h after ZIKV (MOI = 1) or mock infection. CPE of ZIKV is indicated by rounding and detachment of cells. (C) Wild type RD or ERp57-KO cells were infected with ZIKV (MOI = 1). Infectious viral particles in culture supernatants and cell lysates were measured by plaque assay. (D) and (E) U87 cells were transfected with scramble siRNA or siERp57 for 24 h. Cells were infected by ZIKV (MOI = 1) for 24 h. Intracellular RNA samples were collected and used for quantitative RT-PCR (D). Viral titre was determined by TCID₅₀ after the indicated viral infection time (E). (F)-(H) RD or A549 or U87 cells were transfected with scramble siRNA or siERp57 for 24 h. Cells were then infected by ZIKV (MOI = 1) for the indicated time and protein samples were analysed by western blot. (I) Wild type RD cells were transfected with ERp57 plasmid for overexpression. Cells were infected by ZIKV (MOI = 1) with 24 h and protein samples were harvested at the indicated time. Viral titres and qRT-PCR data are means ± SEM from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (unpaired t-test).

Figure 2.

ERp57 depletion inhibits ZIKV-induced ER stress and ROS production. (A) U87 cells were transfected with scramble siRNA or siERp57 for 24 h. Intracellular RNA samples were collected after 24 h infection, later used for quantitative RT-PCR. (B) Wild type RD and ERp57-KO cells were infected with ZIKV (MOI = 1). Intracellular RNA samples were collected at indicated time point, later used for quantitative RT-PCR. (C) RD cells were transfected with scramble siRNA or siERp57 for 24 h. Protein samples were harvested after 48 h ZIKV infection (MOI = 1). (D) Both wild-type and knockout RD cells were infected with ZIKV (MOI = 1) for 48 h and the protein expression were analysed by western blot. (E) Immunofluorescence images showing ROS in WT and KO RD cells. Cells were infected with or without Zika virus (MOI = 1) for 24 h. Cells were stained with ROS (green) and DAPI (blue) and visualized under confocal microscope. (F–I) A549 cells were transfected with either scramble siRNA or siERp57 for 24 h, followed by ZIKV infection for the indicated time. ROS production was stained and measured by confocal image (green) (F), microplate plate reader (G), and flow cytometry (H and I). Results were expressed as mean ± standard deviation (error bars) of at least three repeats. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (unpaired t-test).

Figure 3.

ERp57 depletion decreases ZIKV-induced host cell DNA damage. (A) Representative comet assay images of ZIKV-induced DNA fragmentation. Cells were infected with Zika virus (MOI = 1) 24 h or 48 h before being harvested. Each round dot represents the original cell nuclear area, while the comet shaped tails to the left indicate fragmented DNA. The bigger the DNA tail area or the longer the DNA tail length, the more significant the damage. (B) Immunofluorescence images showing accumulated 8-oxoguanine within WT and KO RD cells. Cells were infected with Zika virus (MOI = 1) 24 h or 48 h before being harvested. Cells were fixed and stained with anti 8-oxoG (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (C) Immunofluorescence images showing foci of gamma H2AX within nucleus of ZIKA infected RD cells. WT and ERp57-KO RD cells were infected with ZIKA virus (MOI = 1) for the indicated time. The cells were fixed and stained with anti-gamma H2AX (red) and ERp57 (green) fluorescence antibody afterwards. (D) Wild type or knockout RD cells were infected with ZIKA virus (MOI = 1). Cell lysate was collected at the indicated time point, and protein expressions were analysed over a time course. (E) and (F) Wild type or knockout RD cells were infected by ZIKV at MOI = 10 for the indicated time. Nuclear and cytosol protein were extracted and separated by Beyotime Kit. The protein expressions were analysed by western blot.

Figure 4.

ERp57 depletion inhibits ZIKV-induced apoptotic cell death. (A) and (B) Flow cytometry analysis of ZIKV-induced early-stage apoptosis. WT or ERp57-KO RD cells were treated with PBS or ZIKV (MOI = 1) dilution 24 h or 48 h before sample collection. Cells treated with hydrogen peroxide (H₂O₂, 0.1 mM) 1 h before collection were used as positive controls. The statistical results of the proportion of healthy (double negative), early apoptotic (Annexin V positive), late apoptotic (double positive) and necroptotic (double positive) cell populations are shown as percentage (B). (C) and (D) and (F) U87 cells were transfected with scramble siRNA or siERp57 for 24 h. Cells were then infected by ZIKV (MOI = 1) for the indicated time. Intracellular RNA samples were then collected and used for quantitative RT-PCR. (E) Both wild type RD cells and knockout cells were infected by ZIKV (MOI = 1) for the indicated time. Intracellular RNA samples were then collected and used for quantitative RT-PCR. (G) and (H) Representative images show the protein expression level of apoptosis related regulators in ZIKV-infected WT or ERp57-KO RD cells. RD cells were infected with ZIKA (MOI = 1), and protein expression analysed over indicated time course. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (unpaired t-test).

Figure 5.

ERp57 is critical for ZIKA nonstructural protein expression and function. (A) Representative immunofluorescent staining images show the localization of ZIKV NS5 (green). WT RD cells were seeded in 24-well plates with pre-coated cover glasses, then infected with ZIKA virus (MOI = 1) for the indicated time. Cells were fixed and stained with anti-NS5 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (B) and (C) Both wild type RD cells and knockout cells were infected by ZIKV (MOI = 1) at the indicated time and protein level was analysed by western blot. (D) Both WT and KO RD cells were infected with ZIKV (MOI = 1) for the indicated time. Cells were fixed and stained with anti-NS1 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (E) Both wild type RD cells and ERp57-KO cell line were transfected with NS1-flag plasmid or vector, then infected with ZIKV (MOI = 1) for 48 h. Protein samples were blotted to investigate the expression level of NS1 and other DDR related factors. (F) Western blot images of WT and ERp57-KO cells transfected with NS1-Flag plasmid or vector for 24 h.

Figure 6.

ERp57 helps NS2B and NS3 binding to promote DDR. (A) Both wild type RD cells and ERp57 knockout cells were transfected by NS2B, NS3 plasmids for 24 h and protein level was analysed by western blot. (B)–(D) Both wild type RD cells and ERp57 knockout cells were infected by NS2B (B), NS3 (C) lentivirus or their combinations (D). The cells were fixed and stained with anti-gamma H2AX (red) and NS2B (green) or NS3 (green). (E) RD cells were seeded in 24-well plates with pre-coated cover glasses, then infected with ZIKA virus (MOI = 1) for 24 or 48 h after cultured for 24 h. Cells were fixed and stained with anti-NS2B (green) fluorescence antibody and NS3 (red) and visualized under confocal microscope, yellow indicates the co-localization of the two proteins. (F) RD cells were infected with ZIKV for 24 h at MOI = 1. The cells were fixed and stained with ERp57 (green) and NS2B (red). (G)–(I) Both wild type RD cells and knockout cells were infected with ZIKV for 48 h at MOI = 1. Whole cell lysates (H) and immunoprecipitated proteins incubated with anti-NS2B antibody (G) were analysed by immunoblotting. GAPDH was used as a loading control for the inputs while IgG was used as the negative control for the co-immunoprecipitation experiments. The densitometric analysis of NS2B and NS3 levels in Figure 6(G), along with the total NS3 level in Figure 6(H), was quantified. The NS2B-bound NS3 was first analysed by NS3 level relative to pulled NS2B from Figure 6(G). Additionally, the ratio of NS2B-bound NS3 to total NS3 from the input sample was further analysed (I). (J) The schematic picture shows the interaction between ERp57 and ZIKV proteins.

Figure 7.

LOC14 decreases ZIKV-induced DNA damage. (A–C) RD cells were treated with the indicated concentrations of LOC14 for 2 h, then infected with ZIKV at an MOI of 1 for 24 h. DMSO was treated in 0 μM group. Intracellular viral RNA level was tested by RT-qPCR (A) and viral protein level was determined by western blot (B). Representative CPE images of RD cells were indicated by rounding and detachment of cells (C). Cells without ZIKV infections are in Figure S7(B). (D) RD cells were treated with different concentrations of LOC14 for 2 h, then infected with ZIKV (MOI = 1) for 24 h. Protein level was further analysed by western blot. (E) RD cells were treated with different concentrations of LOC14 for 2 h, and RD cells were infected with ZIKA virus (MOI = 1) or NS2B and NS3 transfection for 24 h. The cells were fixed and stained with anti-gamma H2AX (red) and Hoechst (blue) afterwards. (F) RD cells were pretreated with LOC14 for 2 h and transfected with NS2B and NS3 for 24 h. Protein levels were analysed by western blot. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (unpaired t-test).