The globoside receptor triggers structural changes in the B19 virus capsid that facilitate virus internalization

Abstract

Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

FIG. 1.

Detection of B19V receptors in UT7/Epo cells by flow cytometry and immunofluorescence. (A) The presence of the B19V receptors Gb4Cer, Ku80, and CD49e (integrin alpha-5) on the surfaces of UT7/Epo cells was determined quantitatively by fluorescence-activated cell sorter (FACS) analysis. The proportion of positive cells is shown in the upper right quadrant of each panel. The background control consisted of cells stained with the conjugated secondary antibody. (B) Immunofluorescence picture of a large field showing UT7/Epo cells (nuclei stained with DAPI) and Gb4Cer (green).

FIG. 2.

Role of cell receptors in B19V attachment. (A) Immunofluorescence scanning microscopy of representative cells showing colocalization of B19V (red) with Gb4Cer or CD49e (green). A merged image is shown. (B) Quantitative comparison of B19V binding to UT7/Epo cells in the presence of antibodies against Ku80, CD49e, and Gb4Cer.

FIG. 3.

Dynamics of B19V receptor detachment and internalization in UT7/Epo cells. (A) At progressive times following incubation at 37°C, total virus (⧫; virus in the supernatant and cell pellet) and cell-detached (▪; virus in the supernatant), cell-associated (▴; virus in the cell pellet), and internalized (○; virus in the cell pellet after trypsinization) virions were quantified. (B) Kinetics of receptor detachment at 37°C (▪) and 4°C (□). The upper, discontinuous line represents the total number of cell-bound virions before incubation. The lower dotted line represents the maximum amount of receptor-dissociated virus at 37°C.

FIG. 4.

Binding and infectivity of native and detached viruses. UT7/Epo cells were incubated with equal amounts of native and detached B19V at 4°C for 1 h. Following washings to remove unbound virus, total DNA was extracted and viral DNA was quantified by quantitative PCR. Virus infectivity was examined by quantification of NS1 mRNA at 24 h postinfection.

FIG. 5.

Analysis of VP1u accessibility and viral DNA exposure following binding to cells. UT7/Epo cells were infected at 4°C with B19V. After being washed to remove unbound virus, the cells were lysed. B19V capsids were immunoprecipitated with MAb 850-55D (against intact capsids) or with a rabbit polyclonal antibody against VP1u. Native particles were immunoprecipitated in the presence of lysis buffer. As a control, an unspecific IgG control antibody was used. (A) Immunoprecipitated capsids were analyzed by SDS-PAGE. (B) Immunoprecipitated virions were quantified by real-time PCR. (C) Quantification of VP1u exposure after prolonged incubation at 4°C for 1 h. (D) Analysis of viral DNA externalization in receptor-bound virions. Following B19V binding to UT7/Epo cells, the cells were washed, lysed, and incubated with DNase I. Subsequently, intact viral DNA was quantified by real-time PCR. As a control for DNase I digestion, capsids pretreated at 65°C for 3 min were used. (E) Infectivity of receptor-detached virus following incubation with DNase I or the AccI restriction enzyme.

FIG. 6.

B19V-globoside interaction in vitro. Increasing concentrations of purified Gb4Cer were incubated with viral particles. Subsequently, capsids were immunoprecipitated with a human MAb against intact capsids (860-55D) or with a rabbit polyclonal antibody against VP1u. Quantification of the immunoprecipitated virions was performed by real-time PCR.

FIG. 7.

Analysis of B19V capsid stability. Similar amounts of various capsid preparations (capsids bound to cells, detached capsids, and capsids from native virus, with or without cells) were exposed to increasing temperatures for 3 min. Following incubation of the capsids with DNase I, total DNA was extracted and quantified.

FIG. 8.

Binding and internalization of native virus and VLPs (with or without VP1). (A) SDS-PAGE of native virus, VLPs (VP1/VP2), and VP2-only particles. (B) UT7/Epo cells were incubated with the different viral particles for 1 h at 4°C, washed intensively, and further incubated at 37°C to allow virus internalization. At increasing times, the cells were washed, fixed, and stained with MAb 860-55D against intact capsids. Alternatively, after 30 min at 37°C, the cells were incubated with trypsin-EDTA to remove uninternalized capsids. Subsequently, the cells were washed, fixed, and stained with MAb 860-55D.

FIG. 9.

Role of the N-terminal part of VP1u in virus internalization. (A) Neutralization activity of two MAbs against B19V. One MAb recognizes intact capsids (MAb 860-55D; α-Caps), and the other recognizes an epitope in the N-terminal part of VP1u (MAb 1418-1; α-N-VP1u). The antibodies were added during (preattachment) or after (postattachment) virus binding or after virus internalization (postinternalization). Virus infectivity was examined by quantification of NS1 mRNA at 24 h postinfection. (B) Capacity of B19V to be internalized in the presence of anti-N-VP1u added after virus binding (postattachment) or during virus binding (preattachment).

FIG. 10.

Schematic representation of the proposed mechanism of B19V binding and internalization in UT7/Epo cells. B19V binds to the Gb4Cer receptor. The binding triggers the exposure of VP1u, which interacts with the coreceptor. However, whenever the interaction with the coreceptor is not possible, the virus detaches from Gb4Cer while keeping VP1u accessible at the surface. Cycles of binding and detachment are repeated until the interaction with the coreceptor occurs, after which the virus is internalized.