Productive cell infection by the prototype strain of CVA21 is mediated by discrete interactions with surface-expressed DAF and ICAM-1 (33
). In this relationship DAF functions to sequester CVA21 to the cell surface for subsequent interactions with ICAM-1 that induce capsid conformational changes and cell entry (34
). However, the question as to whether multiple in vitro cell passages contribute to this pattern of receptor usage or not is a subject of much contention. In particular, the DAF-binding phenotype was an area of much conjecture, considering that the phylogenetically related prototype group A coxsackieviruses A13, A15, A18, and A20, which also use ICAM-1 as a cell internalization receptor, do not bind to surface-expressed DAF (23
). Investigations were thus undertaken to observe whether the DAF-binding phenotype of the CVA21 prototype strain Kuykendall was conserved in low in vitro-passaged clinical isolates of CVA21 or was simply an artifact of multiple passage in cell cultures.
The radiolabeled virus-binding assays described here indicate that three clinical isolates of CVA21 showed receptor attachment patterns similar to that of the prototype CVA21 strain (Kuykendall), patterns characterized by the capacity to bind independently to either DAF or ICAM-1 (Fig. ). The inability of a MAb blockade directed against either DAF or ICAM-1 to completely inhibit virus binding (Fig. ) indicates that both receptors play an essential role in the attachment-infection process of clinical isolates of CVA21. Studies of CVA21 binding in environments of high-level coexpression of DAF and ICAM-1 demonstrate a reduced degree of virus binding compared to environments in which the two receptors are expressed individually (Fig. ). These findings suggest that high-level coexpression of multiple receptors may indeed be inhibitory to optimal lytic infection. It is possible that the close proximity of DAF and ICAM-1, when coexpressed on a host cell surface (34
), results in steric hindrance, causing a reduction in the availability of the receptor-binding sites. If this is the case, it can be reasoned that, whereas high-level expression of both receptors on a host cell does not necessarily correlate with an increased attachment level, an environment with dissimilar expression levels of the two different cellular receptors for the one virus may be potentially advantageous. Such an environment is likely to occur on the mucosal surface of the human enteric tract, where DAF expression is ubiquitous (25
) and at significantly higher levels than ICAM-1, whose endogenous expression level is relatively low (42
), awaiting induction by appropriate cytokines (40
An unexpected finding of the present study was the capacity of low-passage clinical CVA21 isolates to utilize DAF interactions in a more functional role by of lytically infecting RD cells solely via DAF binding in the absence of antibody cross-linking (Fig. ). A possible explanation for these novel findings is that the virus capsids of the clinical CVA21 isolates are able to cross-link DAF in a more substantial fashion than the prototype strain, thereby permitting virus internalization in a mechanism similar to the artificial cross-linking action of anti-DAF MAbs (Fig. ). Similarly, differences in receptor usage have been observed between CVB3 prototypes and low-passage clinical isolates (4
). More recently, variation in the utilization of different αv
integrins has been reported between laboratory and field strains of foot-and-mouth disease virus, demonstrating that virus isolates can exhibit altered affinities for their cellular receptors (10
Here we confirm that, in the absence of ICAM-1, MAb cross-linked DAF can serve as a functional internalization receptor for both prototype and clinical CVA21 strains (Fig. ). It has previously been proposed that entry of CVA21 mediated by MAb cross-linked DAF occurs via caveolae, in contrast to the clathrin-coated pit entry route used during virus interaction with ICAM-1 (31
). CVA21 entry via caveolae containing the cross-linked DAF hypothesis is supported by evidence indicating that MAb clustered DAF is endocytosed after recruitment into caveolae (20
). A possible role for DAF interaction in caveola-mediated CVA21 entry has been confirmed by recent reports of cell internalization of a DAF binding strain of EV11 via lipid rafts and/or caveolae (37
The widespread expression of DAF throughout the mammalian body (24
) offers an adaptive advantage to viruses that display a higher affinity for DAF and can utilize this receptor for internalization. Such viruses may have an increased pathogenicity compared to other strains due to the expression of DAF on erythrocytes (16
), offering DAF-binding viruses a readily available vehicle for travel throughout the human body. Interestingly, serial passage of coxsackievirus B3 and B5 isolates in polarized epithelial cells (where their natural internalization receptor, the coxsackie and adenovirus receptor [3
], is located in tight cell-cell junctions and DAF on the apical surface) selected for DAF-binding variants, suggesting an important role for DAF infection of epithelial cell mucosal surfaces (36
Genetic analysis of the P1 region of the genome coding for the capsid structural proteins detected a number of differences in the deduced amino acid sequences between clinical CVA21 isolates and the prototype strain. None of the observed coding changes mapped to the previously determined ICAM-1 footprint (44
), and the differences were scattered throughout VP1, VP2, and VP3. Residues constituting the ICAM-1 footprint were conserved in both the prototype Kuykendall strain and all clinical isolates, except for amino acid 168 in VP2 (Fig. ). At position 168 of VP2 the amino acid substitution (Val to Ala) is conservative and potentially of little significance in the conformation of the ICAM-1 binding site. Clinical isolate 272101, which exhibited significant lytic activity in RD cells expressing DAF only (although the lytic activity was not as great as that of the remaining clinical isolates), possessed 13 of 14 coding changes observed between all clinical isolates with respect to the prototype stain. The presence of a further nine additional changes in isolate 272101 compared to the other clinical isolates may have exerted some type of suppression on the enhanced DAF usage phenotype exhibited by isolates 275238 and 2727598. Repeated in vitro cell passage of the clinical CVA21 isolates possessing the elevated DAF usage phenotype in environments of both high DAF and ICAM-1 may exert pressure for the bioselection of virions with enhanced ICAM-1 usage at the cost of reducing functional DAF interactions. Generation of such populations of virions may yield the identification of key P1 amino acid changes responsible for the altered receptor usage phenotypes.
A possible explanation for the reduced DAF usage of isolate 272101 has been provided by a study in which bioselected EV11 variants that had lost their DAF binding phenotype were found to possess specific amino acid changes in the BC loop of VP1 and in the puff region of VP2 (38
). Our sequence analysis revealed the presence of such unique differences in the BC loop of VP1 and the puff region of VP2 of isolate 272101 but not in same capsid region of the other CVA21 clinical isolates (Fig. ). Although not shown to be conclusive in the area of virus attachment and cell entry, these observed 13-amino-acid changes between all clinical isolates and the prototype may potentially play a role in the development of the enhanced DAF usage phenotype. However, whereas not addressed in the present study, the involvement of additional changes located at other regions of the viral genome (e.g., 5′-untranslated region) in mediating cell lytic infection cannot be discarded.
A significant difference, however, between the DAF-EV7, DAF-CVB3 interaction and the DAF-CVA21 interaction is that DAF SCRs 2, 3, or 4 are involved in EV and CVB binding (6
), whereas DAF SCR 1 is involved in CVA21 attachment (33
). Given the overall structural similarities between the EV7, CVB3, and CVA21 capsids, we propose that the involvement of the N-terminal domains of two separate receptors with their own separate binding sites on the CVA21 capsid (i.e., DAF and ICAM-1) may occur at any stage during infection. However, the involvement of SCRs 2 to 4 of DAF in EV7/CVB3 binding (5
) suggests that interactions with additional receptors, such as CAR in the case of CVB3 (3
), may occur after those of DAF in order to minimize the interference with access to the specific DAF-binding epitopes on the virus capsid (14
). Of some interest may be the detection of a slight difference in the migration of VP1 of the prototype strain relative to the clinical isolates able to lytically infect ICAM-1-negative RD cells (data not shown). Similarly, a variant of a CVB3 prototype (CB3-RD), generated after serial passage in RD cells, exhibited an altered VP1 mobility to the prototype and correlated with an altered receptor specificity toward DAF compared to the parental strain (29
Taken together, the results in the present study indicate that the overall binding capability of clinical isolates to their cellular receptors has been conserved with respect to the prototype strain. However, there appear to be some discrete differences in the capacity of clinical CVA21 isolates to utilize these receptors. Similar to CVB3 field strains, the clinical CVA21 isolates possess a phenotype that facilitates the increased use of DAF in cell lytic infection, most probably as a result of passage in humans. The capacity of CVA21 to utilize both DAF and ICAM-1 for attachment and/or infection of host cells suggests the conservation of an advantageous phenotype, allowing individual and/or multiple receptor usage, thereby extending the tissue tropism of the virus and significantly increasing the chances of productive infection.