Expression and folding of human very-low-density lipoprotein receptor fragments: neutralization capacity toward human rhinovirus HRV2.
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.
Ronacher
B
B
Vienna Biocenter (VBC), Institute of Medical Biochemistry, University of Vienna, Dr. Bohr Gasse 9/3, Vienna, A-1030, Austria.
Marlovits
T C
TC
Moser
R
R
Blaas
D
D
D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012229:Rhinovirus / Q000502:physiology*
D017931:DNA Primers; D010446:Peptide Fragments; D011973:Receptors, LDL; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C036053:VLDL receptor