Human FcRn Is a Two-in-One Attachment-Uncoating Receptor for Echovirus 18

Figures

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  FIG 1. FcRn, but not CD55, is an essential receptor for E18, E2, and E15. (A, E, G, and I) Virus titers in the supernatants of HEK293T wild-type (293T WT), 293T FCGRTKO, 293T B2MKO, and 293T CD55KO cells infected with E18-HeB15-254 (A), E18-WF277 (E), E2 (G), and E15 (I). The TCID50 was determined in RD cells at 96 h postinfection. Data depict the means with standard errors of the means (SEM) for triplicates (n = 3). (B, F, H, and J) Growth curve analyses of E18-HeB15-254 (B), E18-WF277 (F), E2 (H), and E15 (J) in 293T WT, FCGRTKO, B2MKO, and CD55KO cells. Data depict the means with SEM for triplicates (n = 3). (C) Light microscopy images of 293T cell lines infected with E18. 293T WT, 293T FCGRTKO, 293T B2MKO, and 293T CD55KO cells were used. (D) Phylogenetic analysis of enterovirus species B based on VP1 gene sequences. Significance was evaluated by using an unpaired two-tailed t test with Welch’s post hoc correction. #, undetected. ****, P < 0.0001; ns, not significant.

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  FIG 2. FcRn is a fundamental receptor for E18 infection. (A) Schematic of E18 receptor CRISPR/Cas9 screening in RD cells. (B) Enrichment of gene expression after E18 infection versus an uninfected control. Genes were rank ordered according to the robust rank aggregation (RRA) scores calculated by MAGeCK. (C) Viral titers in the supernatants of cell lines infected with E18. 293T WT cells, 293T FCGRTKO cells, 293T B2MKO cells, 293T FCGRTKO cells complemented with lentivirus expressing FCGRT, and 293T B2MKO cells complemented with lentivirus expressing B2M were used. (D) Viral titers in the supernatants of wild-type and FCGRT- and B2M-coexpressing CHO cell lines. TCID50 data were calculated by the Reed-Muench method. Experiments were repeated three times. #, undetected. ****, P < 0.0001 (by an unpaired two-tailed t test with Welch’s post hoc correction [C and D]).

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  FIG 3. Direct binding between FcRn and E18 viral particles. (A and B) SPR assay characterizing the binding between E18 full particles and FcRn (A) or CD55 (B). The KD values were calculated using Biacore 3000 analysis software (BIAevaluation version 4.1). (C) Soluble FcRn inhibits E18 infection dose dependently. CAR was used as a control. (D and E) Binding (D) and endocytosis (E) of E18 in green fluorescent protein-expressing CHO-GFP or human FcRn-expressing CHO-FcRn cells. Data depict the means with SEM (n = 3). **, P < 0.01; ***, P < 0.001 (by an unpaired two-tailed t test with Welch’s post hoc correction).

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  FIG 4. Cryo-EM structure of E18 and its complex with receptor FcRn. (A to C) Density maps of full E18 particles at pH 5.5 (A) and E18 in complex with FcRn at pH 7.4 (B) or pH 5.5 (C). (D) Closeup view of the hydrophobic pocket and the pocket factor inside. (E and F) Collapsed pocket without the pocket factor. The density maps are colored by radius, as shown in the key in panel C. Atomic models of the protein are shown in ribbons, and VP1, VP2, VP3, and FcRn heavy chains are shown in blue, green, red, and orange, respectively.

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  FIG 5. The molecular mechanism of the E18 interaction with FcRn. (A) Atomic model of the E18/FcRn complex in the main contacting interface. (B) Details of the interaction between E18 and FcRn. The proteins are colored by chain as shown in Fig. 4. (C) Alignment of VP1 protein from free E18 particles and the E18/FcRn complex at pH 7.4 or pH 5.5. (D) Residues that played a key role in pocket factor release and pocket collapse.

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  FIG 6. FcRn-decorated liposomes facilitate E18 uncoating. (A and B) Negative-staining EM of purified E18 (A) and nickel-charged liposomes (B). (C) E18 was incubated with FcRn-decorated liposomes at pH 7.4. Red arrows indicate the particles attached to the liposomes’ surface. (D) E18 was incubated with FcRn-decorated liposomes at pH 5.5. Green arrows indicate the particles released from the liposomes’ surface.

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  FIG 7. Model for the entry of certain echovirus serotypes into the host cell. The structural proteins VP1, VP2, VP3, and VP4 are shown in dark blue, dark green, light green, and purple, respectively. The viral RNA genome is indicated in orange. The FCGRT domain and B2M domain of FcRn are shown in pink and red, respectively.