Mechanism of Action and Capsid-Stabilizing Properties of VHHs with an In Vitro Antipolioviral Activity

Abstract

Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC 50 s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a ThermoFluor thermal shift assay, in which the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid is determined, either in the presence or in the absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (cryo-EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating. IMPORTANCEThe study describes the mechanism of neutralization and the capsid-stabilizing activity of five single-domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight into the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

“…One of the most direct mechanisms is blocking the receptor binding sites. Such neutralizing mAbs and Nanobodies were previously identified for influenza virus, HIV, herpes simplex virus, rhinovirus, and others [35][36][37][38][39][40][41]. For example, in the case of HIV, with the aid of an extra long CDR3 loop, the neutralizing Nanobody D7 effectively competed for the CD4 binding site on gp120 protein [42].…”

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

“…The selection, production, and purification of the VHHs were previously described in detail (4,5). Originally, the VHHs used in these experiments were obtained from a dromedary immunized against poliovirus type 1.…”

“…The VHH-encoding cDNA was cloned into the phagemid vector pHEN4 (2) for phage display panning on immobilized poliovirus type 1 and recloned into vector pHEN6(c) for expression in Escherichia coli WK6 cells. Finally, the VHHs were purified as described previously (5) and stored at Ϫ80°C in Tris buffer (137 mM NaCl, 25 mM Tris, pH 7.2). Five different recombinant VHHs were used: PVSP6A, PVSS8A, PVSP19B, PVSS21E, and PVSP29F.…”

“…Motivated by searches for potential antiviral agents, VHHs that specifically bind to and neutralize poliovirus type 1 were recently selected and identified (4) and were further characterized (5,6). In a previous publication (5), we showed that the binding footprints of two VHHs (PVSP6A and PVSP29F) on the poliovirus surface overlap extensively with the footprint of the poliovirus receptor (PVR; also called CD155) and that they produce well-ordered icosahedrally symmetric complexes.…”

“…In a previous publication (5), we showed that the binding footprints of two VHHs (PVSP6A and PVSP29F) on the poliovirus surface overlap extensively with the footprint of the poliovirus receptor (PVR; also called CD155) and that they produce well-ordered icosahedrally symmetric complexes. It was also shown that the mechanisms of neutralization operate at multiple stages of the infection process and that all of the neutralizing VHHs stabilize the virus to prevent viral expansion (5), which is an essential step in the infection of cells.…”