Binding of B19V to the cellular receptor globoside (Gb4Cer) induces structural changes in the capsid leading to accessibility of the N-terminal region of VP1 (VP1u). Although such large receptor-induced structural changes have not yet been observed in other parvoviruses, a slight opening of the 5-fold axis pore has been detected following binding of adeno-associated virus 2 (AAV-2) to heparin (22
). The expansion of the 5-fold axis pore is believed to facilitate the externalization of VP1u, which in all parvoviruses studied so far occurs during the intracellular trafficking of the capsid mediated by the acidified endosomal environment (18
). Therefore, B19V would be unique among parvoviruses in that the conformational changes leading to the accessibility of VP1u sequences do not occur inside the cell but prematurely, at the cell membrane. The question remains whether VP1u is not accessible because it is buried within the capsid, similar to those of other parvoviruses, or because it is already external but not accessible to antibodies due to a particular “unattainable” conformation. An argument against the latter hypothesis is that the 5-fold channel, which is suggested to be used during VP1u externalization, is closed in the B19V particle (1
). However, it cannot be excluded that structural rearrangements during infection, similar to those observed in AAV-2 capsids bound to heparin (22
), may eventually open or expand a flexible pore. It is also not known whether the observed rearrangements concern the complete VP1u region or some domains. In our previous studies, we examined the accessibility of two distant regions of VP1u: one region in the N-terminal part, which contains numerous neutralizing epitopes, and a second region in the opposite part, where the PLA2
motif is located. Both regions were not accessible to antibodies in the native particles but became exposed upon mild heat and low-pH treatments without capsid disassembly (3
The early VP1u exposure prior to virus internalization and the fact that it remains stably exposed in receptor-dissociated virus would explain how originally inaccessible regions of the capsid can harbor numerous neutralizing epitopes (2
). Accordingly, neutralizing antibodies raised against those epitopes would not target circulating native viruses but would exclusively target viruses in the process of cell attachment and receptor-dissociated viruses.
Similar to the case with MVM (10
), physicochemical treatments of B19V capsids in vitro
lead simultaneously to both VP1u and viral genome exposure without capsid disassembly (29
). However, an important difference is that receptor binding leads to VP1u exposure but not to viral genome externalization (Fig. ). Therefore, physicochemical treatments in vitro
do not entirely mimic or are not able to fully dissect the sequential structural transitions of the virion during the process of cell entry. Unexpectedly, VP1u exposure did not facilitate the externalization of the viral genome in response to increasing temperatures (Fig. ). On the contrary, receptor-bound capsids were remarkably more resistant than native or detached capsids. Increased capsid rigidity might assist incoming viruses during entry. In our previous studies, we showed that the B19V genome, in contrast to other parvovirus genomes, can easily be externalized upon mild acidification similar to that found in endosomes/lysosomes (29
). Therefore, the natural resistance of B19V capsids would apparently be insufficient to prevent the externalization of the viral DNA in the acidic endosomal environment. Our results indicate that following binding to globoside, alone or in concert with other molecular structures at the cell surface, B19V acquires a remarkably superior stability. The increased resistance, in contrast to VP1u exposure, was reversible upon receptor detachment (Fig. ). In this way, B19V capsids would become more resistant when needed but then become flexible again upon receptor dissociation and endosomal escape to facilitate uncoating.
It is reasonable to expect that receptor-induced conformational changes are necessary for subsequent interaction events leading to virus internalization. In line with this assumption is the fact that VP2-only capsids, lacking VP1 (Fig. ), were unable to be internalized (Fig. ). Moreover, an antibody (MAb 1418-1; referred to as anti-N-VP1u) recognizing an epitope in the N-terminal part of VP1u (aa 32 to 40) (12
) blocked B19V infectivity and hampered virus internalization, but only when present during binding, not when added after virus binding (Fig. ). This result suggests that the externalized N-terminal part of VP1u is engaged in binding events required for internalization, preventing antibodies from binding due to ligand occupancy. Since anti-N-VP1u could only partially block internalization, the epitope recognized by the antibody (aa 30 to 42) might not be the region directly implicated in binding events. Growing evidence indicates that α5β1 integrin might be required for virus internalization (36
). The α5β1 integrin binding motif RGD is present in the N-terminal part of VP1u regions from rhesus (aa 141 to 143) and simian (aa 139 to 141) erythroviruses and aligns with RGE and RGA motifs in these regions from B19V (aa 104 to 106) and pig-tailed erythrovirus (aa 106 to 108), respectively. While RGD is the classical α5β1 integrin binding motif, RGE and RGA motifs have also been found to interact specifically with α5β1 integrin (7
). Although the mere presence of RGD or RGD-like motifs does not warrant an interaction with integrin, the fact that these sequences are conserved in all members of the Erythrovirus
genus is rather suggestive. Interestingly, this region is adjacent to the epitope recognized by anti-N-VP1u (aa 30 to 42). It was previously shown that B19V infection was not inhibited by RGD-containing peptides (36
), raising the possibility that B19V-integrin interaction differs from those involving classical RGD motifs. In order to elucidate this question, further experiments are in progress to examine binding of native and mutated VP1u domains with integrin subunits.
Previous studies demonstrated that B19V does not bind to isolated Gb4Cer (20
), suggesting that B19V binds Gb4Cer at the cell surface as part of a complex involving other structures. However, incubation of B19V particles with increasing concentrations of purified Gb4Cer triggered changes in the original inaccessible configuration of VP1u, which became increasingly accessible on the capsid surface (Fig. ). A possible explanation for our results is that binding of B19V to isolated Gb4Cer is highly unstable due to the lack of required stabilizing coreceptors. This might also explain the detachment of a large proportion of cell-bound particles observed in this study (Fig. ). Interestingly, reattachment of receptor-dissociated B19V to the receptor from which it detached was not detectable at 4°C and occurred only after several hours at 37°C, probably with newly expressed and/or recycled receptors (Fig. ). In contrast, when the detached virus was added to uninfected cells, these particles bound with a higher efficiency and were more infectious than the native virus (Fig. ). This observation might represent a strategy to avoid repetitive “abortive” attachments to the same receptor and in this way increase the probability of productive bindings leading to virus internalization.
An understanding of the events preceding virus internalization is critical for establishing strategies for the prevention and treatment of B19V infections. Our results suggest a complex internalization mechanism involving various steps in a sequential manner, as illustrated in Fig. . B19V binds initially to Gb4Cer, which triggers changes in the VP1u conformation. The structurally altered capsid is ready for a subsequent interaction, which involves binding of the N-terminal part of VP1u with the coreceptor. However, in situations where interaction with the coreceptor is not possible, the virus detaches from Gb4Cer. The procedure is repeated until the second interaction is possible, and virus internalization ensues as a consequence. The mechanism of “detachment-reattachment” would enhance the infection by minimizing the consequences of abortive attachments that do not lead to virus internalization.
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 (more ...)