Abstract
The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.
Highlights
Echovirus 11 (EV11)1 is a member of the genus Enterovirus, family Picornaviridae
Our results indicate that DAF binds to EV11 with a low affinity and very fast kinetics and that most of the binding energy is contributed by the DAF short consensus repeats (SCR) domain 3
Binding of recombinant DAF to EV11 was measured by injecting the proteins, such as DAF234, through a BIAcore flow cell with EV11 covalently coupled to the sensor surface as well as through a mock-coupled control flow cell in which the sensor surface had been subjected to the coupling reaction in the absence of EV11
Summary
The expression of soluble DAF in the yeast P. pastoris has been described in detail elsewhere [16]. Debris was removed by low speed centrifugation, and the virus was pelleted through a 30% sucrose layer. We showed that incubation at low pH does not affect virus infectivity (data not shown), indicating that such treatment does not disrupt virion structure. This is not unexpected since the virus replicates in the intestine, having passed through the very low pH environment of the stomach. Different levels (1,300 –16,200 response units or RU) of virus were immobilized by varying the length of the activation step from 30 s to 5 min. All experiments were performed at a flow rate of 40 l/min at 25 °C using as running buffer commercially obtained (BIAcore AB) HEPES-buffered saline (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20)
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