Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.

Preferential recognition of the very low-density lipoprotein receptor ligand binding site by antibodies from phage display libraries.

Screening of a phage library displaying single chain fragments of the variable regions of human immunoglobulins (scFv) for binding to the ovarian chicken very low-density lipoprotein/vitellogenin receptor (OVR) led to the isolation of several antibody fragments with high affinity. As for the natural ligands of OVR, receptor binding of all antibody fragments is strictly Ca(2+)-dependent and is prevented by receptor-associated protein (RAP). Moreover, attachment of human rhinovirus serotype 2 (HRV2) to this receptor is inhibited by all scFvs. In contrast to conventional immunization, the in vitro selection method thus exclusively led to antibodies that attach to or close to the ligand binding site and thereby block the receptor-ligand interaction.

An antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection.

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff = 2 x 10(8) M-1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/alpha 2MR), and it competes for binding of receptor-associated protein to LRP/alpha 2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/alpha 2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.