Characterisation of a secreted MFSD6-Fc microbody as a decoy receptor for respiratory enterovirus D68

Figures

• 

  Fig. 1. Engineered secreted MFSD6 microbody binds to EV-D68. (a) Spatial structure prediction of the MFSD6 protein. The second extracellular domain of MFSD6 is highlighted in red. (b) Schematic diagram of the secMFSD6 microbody (Mb). (c) Schematic illustration of the virion capture assay used to detect the interaction between purified EV-D68 particles and secMFSD6 Mb. (d and e) Purified circulating MO strains (d) or prototype Fermon strains (e) of EV-D68 were incubated with secCtl Mb or secMFSD6 Mb at 4 °C for 1 h. Subsequently, 100 μL of protein G agarose was added and mixed with each sample for 24 h. After washing the samples six times with wash buffer, RNA was extracted for RT-qPCR to detect captured EV-D68 RNA. Data are represented as the mean ± standard deviation (SD) (n = 3). N = 3 biological replicates. Two-way ANOVA (d and e) was used to assess statistical significance.

• 

  Fig. 2. secMFSD6 Mb inhibits receptor recognition by EV-D68. (a)The diagram shows that the binding of EV-D68 to secMFSD6 Mb hinders the binding of virus particles to MFSD6. (b) Co-immunoprecipitation (Co-IP) of MFSD6 with EV-D68 MO viruses in the presence of secCtl Mb or secMFSD6 Mb. (c) Captured viral RNA was detected by RT-qPCR. Data are represented as the mean ± SD (n = 3). N = 3 biological replicates. Welch’s t-test was used to assess statistical significance.

• 

  Fig. 3. secMFSD6 Mb inhibits the attachment and entry process of EV-D68. (a) Schematic of the viral attachment and entry assay. (b–g) EV-D68 viruses were incubated with secCtl Mb or secMFSD6 Mb for 30 min. (b–d) A549 cells were washed with cold PBS before infection and then incubated with treated EV-D68 (Fermon, b; MO, c; KY, d) or secCtl Mb or secMFSD6 Mb at 4 °C for 2 h to facilitate virus attachment. (e–g) A549 cells were washed with prewarmed PBS before infection and then incubated with treated EV-D68 (Fermon, e; MO, f; KY, g) or secCtl Mb or secMFSD6 Mb at 37 °C for 2 h to facilitate virus entry. Data are represented as the mean ± SD (n = 3). N = 3 biological replicates. Welch’s t-test (b–g) was used to assess statistical significance.

• 

  Fig. 4. secMFSD6 Mb destroys the integrity of EV-D68 viral particles. (a) EV-D68 Fermon viruses were incubated with secMFSD6 Mb or secCtl Mb for 1 h. The samples were then subjected to ultracentrifugation through a 20% sucrose cushion at 41,000 rpm for 12 h at 4 °C. Subsequently, three distinct fractions were collected as indicated. (b) RT-qPCR was used to detect the presence of EV-D68 RNA within the three collected fractions, labelled as F1, F2, and F3. (c) The relative proportions of the F1, F2, and F3 fractions, as determined in (b), were quantified and compared. Data are represented as the mean ± SD (n = 3). N = 3 biological replicates. Unpaired t-test (b and c) was used to assess statistical significance.

• 

  Fig. 5. Electron microscopy observation of the effect of secMFSD6 Mb on the integrity of EV-D68 viral particles. (a and b) EV-D68 MO viruses were incubated with secCtl Mb or secMFSD6 Mb for 12 h. Virus particles were observed by transmission electron microscopy (TEM). Scale bars represent 200 nm or 20 nm.

• 

  Fig. 6. secMFSD6 Mb inhibits EV-D68 viral replication in respiratory cell lines. (a–g) EV-D68 MO viruses were incubated with secCtl Mb or secMFSD6 Mb for 30 min. Calu-3 cells, BEAS-2B cells, and A549 cells were infected with treated EV-D68 MO viruses. (a) EV-D68-induced cytopathic effects (CPEs) were inhibited by secMFSD6 Mb. Scale bars equal 20 μm (BEAS-2B cells) or 70 μm (Calu-3 cells and A549 cells). (b–d) The secMFSD6 Mb inhibits viral protein VP1 expression in respiratory cell lines (Calu-3 cells, b; BEAS-2B cells, c; A549 cells, d). (e–g) The secMFSD6 Mb inhibits viral titres in the supernatant of respiratory cell lines (Calu-3 cells, e; BEAS-2B cells, f; A549 cells, g). Data are represented as the mean ± SD (n = 3). N = 3 biological replicates. Welch’s t-test (e) or unpaired t-test (f–g) was used to assess statistical significance.

• 

  Fig. 7. secMFSD6 Mb inhibits EV-D68 viral replication in vitro and in vivo. (a and b) EV-D68 viruses were incubated with secCtl Mb or secMFSD6 Mb for 30 min. Primary human bronchial epithelial cells (HBECs) were infected with treated EV-D68 viruses. The secMFSD6 Mb inhibits viral titres in the supernatant (MO, a; Fermon, b). (c) Neonatal ICR mice (n = 9 per group) were intraperitoneally inoculated with EV-D68 US/MO/14-18947 (1.0 × 107 TCID50) pre-incubated with secCtl Mb or secMFSD6 Mb and monitored daily for 15 days. The corresponding number of animals at risk for each group throughout the time course is indicated in the lower panel. The log-rank (Mantel–Cox) test was employed in this study. Data are represented as the mean ± SD (n = 3). N = 3 biological replicates. Unpaired t-test (a and b) was used to assess statistical significance.