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Echoviruses (EVs), members of the Enterovirus genus of the family Picornaviridae, are small positive-strand RNA viruses with icosahedral symmetry which produce a broad spectrum of diseases in humans (8). Productive cell infection requires the uncoating of the virus particle and release of the RNA genome into the cell cytoplasm. The conversion of the 160S mature virions into 135S A and 80S empty particles (reviewed in reference 14) is considered indicative of this process. Poliovirus A particle formation can be induced by soluble poliovirus receptor (6, 20), whereas soluble decay-accelerating factor, a receptor for a number of enteroviruses including EV12 (17), does not induce EV A particles (12). Moreover, conversion of poliovirus to A particles is rapid compared with the conversion of EV (12). Both uncoating and the thermal stability of virions are thought to be regulated by the presence of "pocket factors" in the hydrophobic pocket at the base of the canyon floor. Known pocket factors are fatty acid (FA) or FA based; poliovirus uses sphingosine (14), coxsackievirus B3 and EV1 both use palmitate (3, 11), and bovine enterovirus uses myristate (15). Enterovirus uncoating can be inhibited by pocket factor mimetics like WIN compounds (10, 14) and by the pocket factor itself (5). The uncoating of EV, but not poliovirus, can also be blocked by normal albumin (18); since this protein contains FAs (7), we considered the possibility that FAs were responsible for this block. However, FA-depleted albumin also blocks EV infection (18). We have now investigated the block on EV infection caused by FA-depleted albumin by using wild-type (WT) EV12 and three thermolabile rhodanine-resistant EV12 mutants (1, 2, 8).

Normal bovine serum albumin (A-7638; Sigma) and FA-depleted albumin (A-0281; Sigma) were solubilized in serum-free Dulbecco modified Eagle medium (DMEM) (final pH 7.4). EV12 and the rhodanine-resistant mutants numbered 9, 17, and 20 were incubated for 1 h at 37°C with normal or FA-depleted albumin (2%, wt/vol), or rhodanine (200 µg/ml). Virus was then diluted in serum-free DMEM and adsorbed to 24-well plates of rhabdomyosarcoma (RD) cells at 95% confluency for 30 min. Cells were incubated in serum-free DMEM, and at 7 h postinfection, cells were assayed for infected cells by using an immunofocal assay (18). Preincubation of WT EV12 or mutants 17 or 20 with normal albumin and rhodanine did not affect infectivity, whereas infection by mutant 9 was enhanced by 180% (Fig. 1). In contrast, infectivity of all the viruses was reduced after preincubation with FA-depleted albumin; WT EV12 was decreased by 50%, and infectivity of the more thermolabile rhodanine mutants (8) was reduced by 70% (mutant 17) to greater than 99% (mutants 9 and 20). The rhodanine mutants map to an internal region next to the threefold axis of symmetry that is known to modulate virion stability (8). Mutants 9 and 17, respectively, have substitutions of V₁₅₇A and V₁₀₁A in VP1, whereas mutant 20 has a mutation of F₅₃Y in VP4. Preincubation with FA-depleted albumin was also found to reduce the infectivity of EV7, but not of poliovirus (data not shown). These results indicate that the reduction in EV infectivity induced by preincubation with FA-depleted albumin is irreversible and that the more thermolabile rhodanine-resistant mutants are more susceptible to FA-depleted albumin mediated inactivation.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   EV12 infection is irreversibly inhibited by FA-depleted albumin. Virus (106 50% tissue culture infective doses/ml) was preincubated with serum-free DMEM containing normal or FA-depleted albumin (2%, wt/vol) or rhodanine (200 µg/ml) for 1 h at 37°C. After dilution in serum-free DMEM, virus infectivity was determined by using a blue-cell immunofocal assay with the anti-enterovirus monoclonal antibody 5-D8/1 (18, 19).

³⁵S-radiolabelled WT EV12 (approximately 40,000 cpm) in 500-µl volumes of serum-free DMEM with or without normal or FA-depleted albumin (2%) was incubated for 0 to 6 h at 37°C. Virus binding to RD cells was then determined on ice (17, 19). Pretreatment of virus with FA-depleted albumin significantly reduced virus binding, whereas pretreatment with serum-free DMEM or normal albumin had no effect (Fig. 2). A similar reduction in virus binding was found for EV7 pretreated with FA-depleted albumin, but not with normal albumin (data not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Pretreatment of WT EV12 with FA-depleted albumin inhibits virus binding. RD cells were incubated on ice for 1 h with 35S-radiolabelled WT EV12 (approximately 40,000 cpm) that had been preincubated in serum-free DMEM with or without normal or FA-depleted albumin (2%, wt/vol) from 0 to 6 h at 37°C. After adsorption for 30 min on ice, cells were washed to remove nonadsorbed counts, and quantity of bound virus was determined by scintillation counting.

The buoyant density of the FA-depleted albumin-treated virus was investigated by sucrose gradient analysis to determine whether the formation of 135S A particles had been induced (Fig. 3). ³⁵S-radiolabelled WT EV12 (approximately 40,000 cpm) in 1-ml volumes of serum-free DMEM containing either normal or FA-depleted albumin (2%) was incubated for 2 h at 37°C. Virus was then sedimented through parallel 15 to 45% sucrose gradients for 4.5 h at 25,000 rpm in a Beckman SW28 rotor, and fractions were quantified by scintillation counting. When treated with normal albumin, the majority of virions remained at 160S. In contrast, when treated with FA-depleted albumin, most of the virus was converted to 135S A particles. Both normal and FA-depleted albumin also generated a smaller peak of 80S empty particles; since the input material was gradient-purified 160S particles, one interpretation is that the 80S particles may be derived directly from 160S particles rather than from a 135S A particle intermediate.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   FA-depleted albumin induces A particle formation of WT EV12. 35S-radiolabelled WT EV12 (40,000 cpm) was incubated at 37°C for 2 h in the presence of either normal or FA-depleted albumin. Virus was then sedimented through parallel 15 to 45% sucrose gradients for 4.5 h at 25,000 rpm in a Beckman SW28 rotor, and the radioactivity in fractions was quantified by scintillation counting.

The results of this investigation suggest that normal and FA-depleted albumin block EV infection by different mechanisms. We suggest FA-depleted albumin probably induces A particle formation by stripping stabilizing pocket factors from mature virions; the greater loss of infectivity of the thermolabile mutants compared with WT EV12 supports this conclusion. Conversely, the increase in the thermal stability of mutant 9 induced by normal albumin (Fig. 1) and the observation that this protein blocks EV uncoating (18) suggest that albumin or the FA associated with it has EV-stabilizing properties.

The induction of EV A particle formation by FA-depleted albumin demonstrates that this process can be promoted by a protein other than the receptor. Furthermore, unlike poliovirus, EV uncoating can be blocked by normal albumin (18), antibodies to 2-microglobulin (reference 19 and unpublished data), and CD59 (4). Taken together, these results suggest that poliovirus and EVs may have different strategies for coupling thermal stability with the associated process of uncoating; these differences may be pivotal in influencing the pathogenic nature of these viruses. It has previously been suggested that a factor(s) other than humoral antibodies may modulate EV-associated meningitis, which is often self-limiting (9, 16), and we have already proposed that the influx of albumin into the cerebrospinal fluid during EV-induced meningitis may prevent further virus replication and host tissue injury (18). Based on the results presented here, we speculate that factors such as age, diet, stress, and diabetesall of which affect the plasma levels of FA (13)may also influence EV pathology by modulating virus dissemination and uncoating.