Once it was established that CVA21 eluted from surface-expressed DAF retains a high level of infectivity (Fig. ), investigations focused on whether DAF-eluted virus could play an active role in the pathogenesis of natural CVA21 infections. The specific question to be addressed was to determine whether virus bound by surface-expressed DAF could initiate a productive infection utilizing a delayed induction of cell surface ICAM-1. In an attempt to simulate such an environment, DAF-expressing RD cells (ICAM-1 negative), normally refractive to CVA21 lytic infection, were transduced to express ICAM-1 or CD36 (using recombinant adenovirus vectors) at 0, 6, and 24 h following initial CVA21 binding to RD cell surface DAF. Flow-cytometric analysis revealed significant levels of surface ICAM-1 expression at 4 h posttransduction, with expression increasing to maximal levels approximately 16 h after adenovirus inoculation (Fig. ). Additional flow-cytometric analysis (Fig. ) and Western blot assays (data not shown) confirmed high-level expression of both ICAM-1 and CD36 at 24 h after transduction of the RD cells by the appropriate receptor-bearing recombinant adenovirus, while levels of endogenous DAF expression in all cells were comparable. Viral infectivity assays were performed to compare the levels of progeny CVA21 propagated in the presence of transduced ICAM-1 and CD36 receptor expression at 0, 6, and 24 h following viral binding to endogenous RD cell DAF. The RD cells transduced to express ICAM-1 at 0, 6, or 24 h after initial inoculation with CVA21 produced significantly higher viral yields (approximately 200-fold) than cells induced to express a mock receptor (CD36) or nontransduced RD cells (Fig. ). Multicycle replication of CVA21 in RD cells transduced to express ICAM-1 at 0, 6, or 24 h after DAF binding resulted in complete lytic destruction of the cell monolayers, whereas no cell lysis was observed in cells expressing CD36 or in nontransduced cells (Fig. ; data not shown). No cell lysis of non-CVA21-infected RD cells transduced with recombinant adenoviruses containing ICAM-1 and CD36 cDNAs was observed (Fig. ).
FIG. 3. CVA21 induced lytic infection of RD cells following delayed induction of ICAM-1 expression. (A) Time course of ICAM-1 expression following adenovirus transduction. RD cells were induced to express human ICAM-1 by transduction with 2.5 × 106 TCID (more ...)
FIG. 4. CVA21-induced lytic infection of RD cells transduced with ICAM-1- or CD36-expressing adenovirus. DAF-expressing RD cells were infected with the prototype strain of CVA21 (multiplicity of infection [moi] = 1.0 TCID50) for 1 h at 37°C. Following (more ...)
Prototype and clinical isolates appear to vary with respect to enteroviral interactions with DAF (3
). In the absence of ICAM-1 expression and antibody-cross-linked DAF, clinical isolates of CVA21, to various degrees, achieve host cell lytic infection, possibly by cross-linking DAF via specific viral capsid interactions (17
). Despite detailed descriptions of DAF interactions for numerous clinical enterovirus isolates, actively defined roles for DAF during lytic infection of prototype enteroviral strains have not been forthcoming. The general consensus from many studies investigating enterovirus-DAF interactions is that DAF functions as a viral concentration receptor, simply accumulating virions on the cell surface for interaction with additional functional internalizing receptors (26
). It has been suggested that the nature of DAF binding to enteroviral capsids is of low affinity because of a very fast dissociation rate constant (13
). In contrast, interactions of ICAM-1 with viral capsids of similar architecture show comparable affinities but with significantly slower kinetics, consistent with binding to a relatively inaccessible site, the capsid canyon (4
Studies addressing the impact of biophysical parameters, such as time, temperature, and pH, on the elution of CVA21 from DAF highlight that CVA21 particles are eluted relatively rapidly from DAF, and this elution is susceptible to increases in temperature and pH (Fig. ). Elution of CVA21 from ICAM-1 is characterized by a dramatic reduction in viral infectivity compared to that of virions eluted from DAF (Fig. ). CVA21 virions eluted from ICAM-1 undergo irreversible capsid conformational changes as a result of receptor binding, leaving them incapable of binding and initiating lytic infection (22
). The capacity of DAF-eluted particles to remain infectious is most probably a result of the inability of DAF to induce CVA21 capsid conformational changes (22
). CVA21 particles eluted from DAF-expressing CHO cells possessed a sedimentation coefficient in sucrose gradients similar to that of infectious 160S particles, whereas CVA21 particles eluted from ICAM-1-expressing CHO cells exhibited a reduced sedimentation coefficient, closer to that of 135S altered particles (data not shown).
The reversible nature of the CVA21 interaction with DAF was highlighted by the capacity of CVA21 to bind to DAF (on ICAM-1-negative cells) and remain in an infectious state for up to 24 h. The retention of infectivity allowed DAF-bound virions to undergo cell entry and subsequent lytic infection when presented with delayed ICAM-1 expression (Fig. and ). In the absence of detectable changes in cell cytopathology, relatively high levels of infectious CVA21 on monolayers of RD cells and RD cells transduced with adenovirus expressing CD36 (Fig. ) persisted throughout the course of investigations, most likely because the residual viral inoculum bound to DAF retained infectivity (Fig. ). Alternatively, it may be due to the presence of a subpopulation of virions within the CVA21 prototype preparation that possess an enhanced DAF usage phenotype, allowing cross-linking of DAF and initiating a subsequent slow infection, a finding previously described for clinical isolates of CVA21 (17
The capacity of the prototype strain of CVA21 to use DAF as an attachment receptor and retain a highly infectious capacity is an extremely advantageous mechanism given the widespread distribution of DAF throughout the mammalian body, particularly on erythrocytes (18
). In this environment, DAF-expressing erythrocytes provide the virus with a ready transport vehicle through the body; bound infectious virus can leave the erythrocyte surface and interact with ICAM-1-expressing cells for lytic infection. It is generally accepted that surface expression of endogenous ICAM-1 throughout the human body is relatively low, awaiting induction by the action of inflammatory cytokines such as tumor necrosis factor alpha and interleukin-1β (30
). During natural human rhinovirus infections, infected cells release such cytokines (20
), which mediate enhanced ICAM-1 expression on surrounding cells. As some low-cell-culture-passage clinical isolates of CVA21 are able to infect cells via DAF alone, the question arises as to whether multiple cell passages have led to the usage of ICAM-1 as an internalizing receptor. The finding that low-passage clinical CVA21 isolates exhibit more rapid and dramatic lytic infection of ICAM-1-bearing cells than those expressing DAF alone (17
) suggests that ICAM-1 usage is most likely not a phenotype acquired through in vitro propagation.
The data presented herein confirm two major roles of DAF during CVA21 lytic infection. First the virus can bind to DAF and elute while still retaining receptor binding capacity and, hence, cell infectivity. Second, CVA21 can bind to DAF and wait for the availability of significant levels of ICAM-1 expression on the same cell or proximal cells to allow viral internalization and subsequent lytic infection. In aspects of both viral evolution and pathogenesis, the capacity to bind to DAF must be viewed as most advantageous for the prototype strain of CVA21 and other DAF-binding enteroviruses in maximizing cell infectivity, a phenotype that is retained and even enhanced in virulent clinical CVA2I isolates.