Expression and Folding of Human Very-Low-Density Lipoprotein Receptor Fragments: Neutralization Capacity toward Human Rhinovirus HRV2

Abstract

Minor group human rhinoviruses (HRVs) use members of the low-density lipoprotein receptor family for cell entry. To investigate the utility of receptor fragments as viral inhibitors, various polypeptide segments derived from the ligand binding domain of human very-low-density lipoprotein receptor (VLDLR) were expressed in a soluble form in bacteria. Whereas none of the fragments was active in virus binding immediately after recovery from the cell lysates, constructs encompassing complement type repeats 1-3, 1-6, and 1-8 spontaneously acquired virus binding activity by incubation at 4 degrees C in buffer containing Ca(2+) ions and lacking any redox system. When immobilized receptor-associated protein (RAP), a specific chaperone for VLDLR, was present during the incubation, the yield of protein active in ligand binding was substantially increased. A VLDLR fragment with repeats 4-6 failed to bind virus; however, it bound RAP. Bacterial expression of truncated VLDLR 1-3 at high yield, easy purification, and folding together with high inhibitory activity toward HRV2 makes this protein a promising starting point for the development of an oligopeptide-based antiviral agent. Using sucrose density gradient centrifugation, we demonstrate the formation of virus-receptor complexes. The recombinant receptors can thus be used for structure determination by electron cryo-microscopy.

Molecular characterization of the first avian LDL receptor

“…However, for antigen production as well as for LA studies, the generation of the entire LA domain of ggLDLR (LA repeats 1-7) inserted into His-tagged MBP provided a valuable tool. In agreement with previous results (27), LA activity of the recombinant protein ( ‫ف‬ 90 kDa, where the contribution of MBP is 42 kDa) was gained by refolding/binding onto immobilized RAP ( Fig. 2A , lanes 3-5).…”

“…Thus, the possibility of testing for the sterol sensitivity of the receptor's expression, the generation of a functionally folded LA domain, and the production of a monospecific inhibitory antibody were key steps in obtaining the current results. Ronacher et al (27) had observed that when bacterially expressed fragments of human VLDLR (LA1-3, 1-6, and 1-8) were incubated in the presence of immobilized RAP, the yield of LA-competent protein was substantially increased. We obtained the same result with the LA domain of ggLDLR (Figs.…”

Species-Specific Receptor Recognition by a Minor-Group Human Rhinovirus (HRV): HRV Serotype 1A Distinguishes between the Murine and the Human Low-Density Lipoprotein Receptor

“…Medium-resolution structures from complexes of the majorgroup viruses HRV14 and HRV16 with soluble ICAM-1 (22,39) as well as of the minor-group virus HRV2 and a soluble fragment of the very-low-density lipoprotein receptor (VLDLR) (14) have been obtained by image reconstruction from cryoelectron microscopy data. Whereas ICAM-1 binds inside the canyon, a cleft encircling the fivefold axes of icosahedral symmetry, the HRV2 receptor complex revealed that the BC loop and the HI loop of VP1 were largely covered by two of the three repeats of the recombinant VLDLR fragment VLDLR [1][2][3] , encompassing ligand binding repeats 1 to 3 only (41).…”

The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding

“…Neutralization tests with antisera and recombinant MBP-V33333 were carried out essentially as described previously (19,26). Briefly, virus at 10 or 1,000 50% tissue culture infective doses as indicated was mixed with twofold serial dilutions of receptor in minimal essential medium containing 2% fetal calf serum, 30 mM MgCl 2 , 100 U/ml penicillin, and 100 g/ml streptomycin (infection medium), incubated for 30 min at room temperature, and transferred onto subconfluent HeLa-H1 cells grown in 96-well plates.…”