Fig 1.
Individual LA3 or LA5 domains of VLDLR LBD can mediate cell attachment and entry of SFV.
(A) Schematic of the ectodomain of VLDLR (left) and LA repeats-Fc fusions (right). LA repeats of VLDLR LBD are numbered. (B) Binding of different LA repeats-Fc fusions, positive control (VLDLRLBD-Fc) or negative control (MXRA8ect-Fc) to SFV virions measured by enzyme-linked immunosorbent assay (ELISA). Mean ± SD of three experiments (n = 6). OD450, optical density at 450 nm. (C) Specific interactions between VLDLRLBD, or different LA repeats and SFV were characterized by SPR. SFV virions were immobilized on the CM5 chip using standard amine coupling chemistry and were then tested for binding with gradient concentrations of VLDLRLBD-Fc or different LA repeats-Fc fusion proteins. The data were analyzed with Biacore 8K Evaluation software (GE Healthcare). KD values are shown as mean ± SEM of three independent experiments. (D) Infection of HEK293T cells with eGFP-expressing SFV (SFV-eGFP) at a multiplicity of infection (MOI) of 5 in the presence of the indicated proteins (100 μg/mL). Cells were imaged by fluorescence microscopy: scale bar, 150 μm. The experiment was performed twice independently with similar results and representative images are shown. (E) Infection of HEK293T cells with SFV-eGFP in the presence of the indicated proteins (100 μg/mL), measured by FACS. Mean ± SD of three experiments (n = 6), One-way ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001; n.s., not significant. (F) VLDLR ectodomain and deletion constructs. LBD LA repeats are numbered. (G) Infection of wild-type K562 cells or K562 cells expressing indicated constructs shown in (F) with SFV-eGFP. Cells were imaged by fluorescence microscopy: scale bar, 150 μm. The experiment was performed twice independently with similar results and representative images are shown. (H) Infection of wild-type or transduced K562 cells with SFV-eGFP measured by FACS. Mean ± SD of three experiments (n = 6), One-way ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001; n.s., not significant.
Fig 2.
Structures of naked SFV virion, SFV in complex with LA3, and SFV in complex with LA5.
(A-C) Left: Three-dimensional reconstructions of naked SFV virion (A), SFV in complex with LA3 (B), and SFV in complex with LA5 (C) viewed along the icosahedral twofold axis. All surfaces are colored radially from blue through white to purple from the lowest to highest radius. One icosahedral asymmetric unit is marked with a white triangle in all reconstructions. Icosahedral symmetry axes are drawn in white and labeled. The white dash line cycles are used to label the block region using in block-based reconstruction algorithm. Right: Thin slices of central sections of the naked SFV virion, SFV in complex with LA3, and SFV in complex with LA5 viewed along the twofold axis. The surfaces are colored the same as the left part. Both LA3 and LA5 are colored red. (D-F) Right: Surface-rendered representations showing cryo-EM density maps of asymmetric units zoned from naked SFV virion (D), SFV in complex with LA3 (E), and SFV in complex with LA5 (F). The E1, E2, capsid of SFV, and LA repeats (LA3 and LA5) are colored white, blue, purple, and red, respectively.
Fig 3.
Detailed contacts between LA repeats and SFV virion.
(A) Ribbon diagrams depicting the structure of LA3 (top) and LA5 (bottom), color-coded in a rainbow gradient from the N-terminus to the C-terminus. Calcium ions are represented as green spheres. All disulfide bonds and the ion-relative residues with negative charges are highlighted. (B) Ribbon-rendered representations showing details of the contacts between bound LA repeats (the left one is LA3 and the right is LA5) and SFV E1-DIII. Residues involved in key salt bridges, hydrogen bonds, and the formation of hydrophobic patches at the interface are shown as sticks. The differences between LA3 and LA5 are highlighted in red. (C) Surface-rendered representations showing the electrostatic potential maps of the binding interface of LA3 (left), LA5 (right), and E1-DIII (middle). Both positive (blue) and negative (red) charged regions involved in LA binding are highlighted. The color of the molecular surface ranges from blue (positive charged) through white to red (negative charged). (D) Surface-rendered representations showing the molecular lipophilicity potential maps of the binding interface of LA3 (left), LA5 (right), and E1-DIII (middle). The color on the molecular surface ranges from dark cyan (most hydrophilic) through white to dark goldenrod (most lipophilic). (E and F) A total of eight LA3 mutants (E) and seven LA5 mutants (F) were tested for their ability to bind to SFV by SPR. SFV was immobilized to 2,500 response units (RU) on a CM5 chip using standard amine coupling chemistry and was then tested for binding with gradient concentrations of LA3 or LA5 mutants. The data were analyzed with Biacore 8K evaluation software (GE Healthcare). KD values are shown as mean ± SEM of three independent experiments. (G) Infection of K562 cells expressing full-length VLDLR, HEK293T cells, or BHK-21 cells with SFV-eGFP bearing E1 mutation at the receptor binding interface. Cells were imaged by fluorescence microscopy. Scale bar, 150 μm. The experiment was performed twice independently with similar results and representative images are shown. (H) Infection of K562-VLDLR cells, HEK293T cells, or BHK-21 cells with the SFV-eGFP mutants by FACS. Mean ± SD of three experiments (n = 6), One-way ANOVA with Tukey’s multiple comparisons test, ****P<0.0001; *P<0.05; n.s., not significant.
Fig 4.
The binding pattern of LA repeats to SFV virion in a twofold axis region.
(A) Three binding modes of LA repeats binding to SFV virion twofold axis region. LA3 binding to all the potential binding sites in the twofold axis region (left), LA5 binding to site-1, -3, -1’, and -3’ in the twofold axis region (middle), and LA2 binding to site-1 and site-1’ in the twofold axis region (right). All the bound LA repeats are shown as red points, and site-1 (or 1’), -2 (or 2’), and -3 (or 3’) are colored cyan, yellow, and hot pink, respectively. (B) Ribbon- and surface-rendered representations showing two LA3s simultaneously binding to adjacent LA repeats binding sites (top: LA3s binding to site-1 and -2; middle: LA3s binding to site-2 and -3; bottom: LA3s binding to site-3 and -1’). LA3s are shown as surface and colored coral red. Site-1 (or 1’), -2, and -3 of SFV twofold axis region are shown in ribbon and colored cyan, yellow and hot pink, respectively. (C) Ribbon- and surface-rendered representations showing the fitting models of two LA5s simultaneously binding to adjacent LA repeats binding sites (top: LA5s binding to site-1 and -2; middle: LA5s binding to site-2 and -3; bottom: LA5s binding to site-3 and -1’). LA5s are shown as surface and colored coral red. Site-1 (or 1’), -2, and -3 of SFV twofold axis region are shown as ribbon and colored cyan, yellow, and hot pink, respectively. The clashes between site-1 bound LA5 or site-3 bound LA5, and site-2 bound LA5 are highlighted. (D) Ribbon-rendered representations showing simulation of two LA2s simultaneously binding to adjacent LA repeats binding sites (top: LA2s binding to site-1 and -2; middle: LA2s binding to site-2 and -3; bottom: LA2s binding to site-3 and -1’). Site-1, -2, -3 (or 3’) of SFV twofold axis region and AlphaFold2 predicted structure of LA2 are colored cyan, yellow, hot pink, and purple, respectively. Residues involved in conformational change are labeled and shown as sticks.
Fig 5.
LA2, LA3, and LA5 synergistically promote cell attachment and entry of SFV.
(A) Binding of individual LA2, LA3, LA5, and LA concatemers to SFV virions measured by ELISA. Mean ± SD of three experiments (n = 6). OD450, optical density at 450 nm. (B) Binding affinity of individual LA2, LA3, LA5, and LA concatemers to SFV virions measured by SPR. KD values are shown as mean ± SEM of three independent experiments. (C) VLDLR ectodomain and deletion constructs. LBD LA repeats are numbered. (D) Infection of wild-type K562 cells or K562 cells expressing indicated constructs shown in (C) with SFV-eGFP. Cells were imaged by fluorescence microscopy: scale bar, 150 μm. The experiment was performed twice independently with similar results and representative images are shown. (E) Infection of wild-type or transduced K562 cells with SFV-eGFP measured by FACS. Mean ± SD of three experiments (n = 6), One-way ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001; ***P < 0.001; **P < 0.01; n.s., not significant.
Fig 6.
The structures of LA3-5 and VLDLR LBD in complex with SFV virion.
(A) Surface-rendered representations showing cryo-EM density maps of twofold axis region from SFV virion in complex with LA3-5 concatemer. The E1, E2, capsid of SFV, and LA repeats (LA3-5) are colored white, blue, purple, and red, respectively. All the LA repeats binding sites, the icosahedral threefold axis (i3), and the quasi-threefold symmetry axis (q3) are labeled. (B) Top: Ribbon-rendered representation showing the model of receptor binding sites in LA3-5 concatemer bound twofold axis region. All the LA repeats binding sites, the icosahedral threefold axis (i3), and the quasi-threefold symmetry axis (q3) are labeled. Bottom: Sideview of ribbon-rendered representation of the area delineated by the red dashed cycle in the top view. The LA repeats binding sites, the icosahedral threefold axis (i3), and the quasi-threefold symmetry axis (q3) are labeled. The membrane inside the SFV virion is shown as a brown fence. (C) Zoom-in view of the ribbon-rendered representation of the area boxed with a black dash box shown at the bottom in panel B. Yellow labels show distances between centers of LAs and their binding sites. The distance between the bound LA and the binding site is measured as 13 Å. (D) Surface-rendered representations showing cryo-EM density maps of twofold axis region derived from SFV virion in complex with low-dose VLDLR LBD. The E1, E2, capsid of SFV and LA repeats are colored white, blue, purple, and red, respectively. All the LA repeats binding sites, the threefold axis (i3), and the quasi-symmetry axis (q3) are labeled. (E) Top: Ribbon-rendered representation showing the model of VLDLR (LA2-5) bound twofold axis region receptor binding sites. All the LA repeats binding sites, the icosahedral threefold axis (i3), and the quasi-threefold symmetry axis (q3) are labeled. (F) Zoom-in view of the ribbon-rendered representation of the area boxed with a black dash box shown at the bottom in panel E, Yellow labels show distances between centers of LAs and their binding site, showing the LA2-5 repeats simultaneously binding to different receptor sites of one twofold axis region. The distance between the bound LA and the binding site is measured as 13 Å.
Fig 7.
The proposed multivalent binding modes accelerated the endocytosis pathway for SFV.
The surface-rendered representation shows SFV utilizes a twofold axis region binding to VLDLR to recognize host cell (A). The binding of VLDLR to the SFV virion occurs in two distinct states within the twofold axis region. Initially, the SFV virion engages LA2 and LA3 concurrently at receptor binding sites 1 and 2 (B, state 1). This action triggers the subsequent attachment of LA5 to receptor binding site-1’ (C, state 2). The binding of LA5 leads to a decrease in the distance between the SFV virion and the host cell, thereby facilitating the attachment of other VLDLRs present on the cell membrane surface to the SFV virion, which tiggers the formation of an endosome and promotes the entry of the SFV virion (D).