GPNMB promotes double-membrane vesicle accumulation and facilitates structural protein transport during PEDV infection

Figures

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  Figure 1.. PEDV infection triggers divergent mRNA and protein expression of GPNMB. (A-C) Vero cells were infected with PEDV (MOI = 1) or mock infected, and the samples were collected at the indicated time points (3, 6, 9, 12, 24, and 36 hpi). (A and B) relative levels of PEDV RNA and GPNMB mRNA were measured by RT-qPCR. (C) Endogenous GPNMB protein was detected by IB. Grayscale values were quantified using ImageJ, normalized to GAPDH, and expressed relative to mock-infected controls (set as 1.0). Data are presented as means ± SEM from three independent experiments. Statistical analysis was assessed by Student’s t-test. **, p < 0.01; ****, p < 0.0001; ns, not significant.

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  Figure 2.. GPNMB promotes PEDV infection in Vero and IPEC-J2 cells. (A to D) Knockdown of GPNMB inhibits PEDV infection in Vero cells. Vero cells were transfected with 50 nM siRNA targeting GPNMB or siNC followed by PEDV (DR13 strain) infection. (A and B) GPNMB mRNA and viral RNA levels were quantified by RT-qPCR. (E and F) endogenous GPNMB and PEDV N protein levels were analyzed by IB. (C and D, G and H) overexpression of GPNMB enhances PEDV infection in Vero cells. Vero cells were transfected with Myc-His-GPNMB or empty vector control, followed by PEDV infection. (C and D) RNA levels were measured by RT-qPCR. (G and H) GPNMB and N protein expression were detected by IB. (I to L) Knockdown of GPNMB inhibits PEDV infection in IPEC-J2 cells. IPEC-J2 cells were transfected with 30 nM sirna targeting GPNMB or siNC for 30 h and then infected with PEDV (HB strain, MOI = 0.01) for 30 h. (I and K) RNA levels were assessed by RT‑qPCR. (J and L) protein levels were assessed by IB. (M to O) Overexpression of GPNMB enhances PEDV infection in IPEC-J2 cells. IPEC-J2 cells were transfected with Myc-His-GPNMB or empty vector for 30 h, then infected with PEDV (MOI = 0.01) for 30 h. (M and N) RNA levels…

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  Figure 3.. Genetic ablation and complementation of GPNMB modulate PEDV infection in Vero cells. (A) Validation of GPNMB knockout, red boxes represent missing sequences. (B to D) GPNMB knockout inhibits PEDV infection. (B) Endogenous GPNMB and viral N protein levels were analyzed by IB. (C) Progeny virus titers were determined by TCID50 assay at 30 h. (D) PEDV infectivity was assessed by immunofluorescence assay (IFA). (E to G) Reintroduction of GPNMB into KO cells rescues PEDV infection. GPNMB-KO cells were transfected with a GPNMB expression plasmid, followed by PEDV infection. (E) GPNMB (0.4ug) and N protein expression were detected by IB. (F) Progeny virus titers were measured in the same manner as in C. (G) PEDV infectivity was analyzed by IFA. Data are presented as means ± SEM from three independent experiments. Statistical analysis was assessed by Student’s t-test. *, p < 0.05; ***, p < 0.001.

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  Figure 4.. GPNMB interacts with the C-terminal domain of the PEDV S1 protein. (A) Endogenous interaction between GPNMB and PEDV S protein. Vero cells were infected with PEDV (MOI = 1) and lysed at 36 hpi. Co-IP was performed using antibodies against GPNMB or PEDV S protein, followed by IB with the indicated antibodies. An isotype IgG was used as a negative control. (B) Subcellular colocalization of GPNMB and PEDV S protein. (upper panel) HEK-293T cells were co-transfected with plasmids expressing GPNMB and PEDV S protein. (lower panel) Vero cells were infected with PEDV. Cells were fixed at 36 h (post-transfection or post-infection) and stained for GPNMB (red) and PEDV S protein (green). Nuclei were stained with DAPI (blue). Images were acquired at 63× magnification. Scale bars, 10 μm. The right panels show the fluorescence intensity of GPNMB and S protein along the lines indicated in the merged images. Representative images from three independent experiments are shown. (C) Direct interaction between GPNMB and PEDV S1. HEK-293T cells were co-transfected with plasmids expressing Myc-His-tagged GPNMB and Fc-tagged PEDV S1 for 36 h. Cell lysates were subjected to co-IP using protein A/G…

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  Figure 5.. GPNMB is dispensable for PEDV attachment and internalization. (A to D) GPNMB knockout or knockdown does not affect PEDV attachment. (A-C) GPNMB-KO or WT Vero cells were incubated with PEDV (DR13 strain, MOI = 5) at 4°C for 2 h. Unbound virus was removed by washing with ice-cold PBS. (A) Cell-associated viral RNA was quantified by RT-qPCR. (B) Attached virions were detected by immunofluorescence staining for the S protein (green). Total fluorescence intensity was quantified using ImageJ. Scale bars, 10 μm. (C) Cell-bound infectious virus was titrated by TCID50 assay after freeze-thaw lysis. (D) IPEC-J2 cells transfected with GPNMB-targeting siRNA or siNC were incubated with PEDV (HB strain, MOI = 2.5) at 4°C for 2 h, washed, and cell-associated viral RNA was measured by RT-qPCR. (E to H) GPNMB knockout or knockdown does not affect PEDV internalization. (E-G) After the 2 h attachment period at 4°C, cells were shifted to 37°C for 1 h to allow internalization, followed by washing to remove non-internalized virus. Internalized virus was quantified by RT-qPCR (E, G) or TCID50 assay (F). (H) Vero cells were infected with PEDV (MOI = 5) as above, then shifted to 37°C for 30 min. Cells…

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  Figure 6.. GPNMB is required for the early replication phase of PEDV. (A to C) Determination of the first life cycle of PEDV. Vero cells were infected with PEDV (MOI = 1) for 1 hpi at 37°C, washed, and then incubated in fresh medium. (A) Intracellular S and N protein levels were analyzed by IB at the indicated time points up to 12 hpi. (B) Intracellular viral RNA levels were quantified by RT-qPCR every 2 h until 12 hpi (C) Supernatants were collected hourly from 6 to 12 hpi and used to infect fresh Vero cells, and infectivity was assessed by immunofluorescence staining for the S protein (green) at 36 hpi. Nuclei were stained with DAPI (blue). Scale bars, 50 μm. Representative images are shown. (D to F) GPNMB knockout impairs early PEDV replication in Vero cells. GPNMB-KO and WT Vero cells were infected with PEDV (MOI = 1) for 7 h, washed, and harvested. (D) Intracellular viral RNA was quantified by RT-qPCR. (E) Cell‑associated virus titers were determined by TCID50 assay after freeze-thaw lysis. (F) Viral N and S protein expression was analyzed by IB. (G and H) GPNMB knockdown impairs early PEDV replication in IPEC‑J2 cells. IPEC-J2 cells transfected with GPNMB-targeting siRNA or siNC were…

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  Figure 7.. GPNMB is required for PEDV-induced DMV accumulation. (A) Colocalization of GPNMB with dsRNA. Vero cells were infected with PEDV (MOI = 1) for 4 h and stained for GPNMB (red) and dsRNA (green). Nuclei were stained with DAPI (blue). The right panels show the fluorescence intensity of GPNMB and dsRNA along the black line indicated in the merged micrograph. Scale bar, 10 μm. (B and C) GPNMB knockout reduces DMV biogenesis. (B) Representative transmission electron microscope (TEM) images of WT and GPNMB-KO Vero cells infected with PEDV (MOI = 1) for 4 and 6 h. DMVs are indicated by red asterisks. L, stacked whorl; N, nucleus; M, mitochondria; E, endoplasmic reticulum. Scale bars = 5 µm (left panels, overview), 500 nm (right panels, magnified views of boxed regions). (C) Quantification of DMVs per cell section at 4 h (n = 15 cells) and 6 h (n = 25 cells) post-infection. (D) impaired viral RNA synthesis in GPNMB-KO cells. WT and KO Vero cells were infected with PEDV (MOI = 1) for 4 h. Intracellular levels of genomic RNA (gRNA) and N mRNA were quantified by RT-qPCR. Data are presented as means ± SEM from three independent experiments. Statistical analysis was assessed by Student’s…

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  Figure 8.. GPNMB promotes the expression and ER-to-Golgi transport of PEDV S and N proteins. (A to D) GPNMB-KO Vero cells were co-transfected with fixed amounts of S- or N- expression plasmids together with increasing concentrations of a GPNMB expression plasmid for 36 h. (A and B) mRNA levels of GPNMB and the N/S genes were quantified by RT-qPCR. (C and D) protein levels were analyzed by IB. (E to H) GPNMB facilitates the redistribution of S and N proteins from the ER to the Golgi.GPNMB‑KO cells were co‑transfected with GPNMB and either S‑ or N‑expression plasmids (total plasmid amount scaled up proportionally for 100‑mm dishes). After 36 h, ER and GA fractions were isolated using a commercial enrichment kit. Enriched proteins were analyzed by IB using antibodies against the indicated tags (His/Flag/HA). Calnexin (ER marker) and GM130 (GA marker) served as loading controls for the respective fractions. (E and F) GPNMB expression reduced S protein levels in the ER while increasing them in the GA. (G and H) a similar but less pronounced trend was observed for N protein, with a modest decrease in the ER and a corresponding increase in the GA. (I and J) GPNMB facilitates the redistribution of…