A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the a2-macroglobulin receptor/LDLRrelated protein (a2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptorassociated protein (RAP), providing evidence that a2MR/LRP also acts as a minor group HRV receptor.Common colds most frequently arise through infection with human rhinoviruses (HRVs). The 102 antigenically distinct serotypes are divided into two groups based on receptor specificity (1, 2). The major group binds to the intercellular adhesion molecule 1 (ICAM-1) (3)(4)(5), and the minor group has been shown to attach to a membrane protein with a relative molecular mass of about 120 kDa (6, 7). ICAM-1 and the poliovirus receptor (8) are members of the immunoglobulin superfamily. As the three-dimensional structures of representative HRVs from the two different receptor groups (9, 10) and of poliovirus (11) show considerable similarity, it might have been expected that the minor group receptor would also belong to this family. However, in this communication we present evidence that minor-group HRVs gain access to the cell via members of the low density lipoprotein (LDL) receptor (LDLR) family (12,13). MATERIALS AND METHODSPurification of HRV2-Binding Protein. Two hundred liters of HeLa cell culture supernatant were concentrated ten times by ultrafiltration, dialyzed against 250 liters of H20 containing 0.02% NaN3, and adjusted to contain 20 mM N-methylpiperazine hydrochloride (pH 4.5). Precipitated material was removed, and the filtered supernatant was applied to a 0.5-liter Macroprep 50 Q column (Bio-Rad). Bound material was eluted with the same buffer containing 0.5 M NaCl. After adjustment to pH 7.2 with 1 M Tris HCl (pH 8), the material was loaded onto a 100-ml Lens culinaris lectin column (Pharmacia), and bound protein was eluted with phosphatebuffered saline (PBS) containing 0.5 M a-D-methyl glucopyranoside and precipitated with (NH4)2SO4 at 50o saturation. The precipitate was dissolved in 200 ml of PBS, the solution was passed over a 40-ml Jacalin agarose column (Vector Laboratories), and bound protein was eluted with 120 ml of 0.1 M a-D-methyl galactopyranoside in PBS and precipitated with (NH4)2SO4 as above. The precipitate was dissolved in 20 mM N-methylpiperazine hydrochloride (pH 4.5) and desalted on a PD-10 column (Pharmacia). Protein was applied onto a Mono Q HR 5/5 column (Pharmacia) and eluted with a gradient of 0-0.5 M NaCl in the same buffer. The binding activity was monitored throughout the purification procedure on ligand blots (7). Active fractions were concentrated to 1.5 ml with a Centricon-30...

The crystal structure of the 2A proteinase from human rhinovirus serotype 2 (HRV2-2A pro ) has been solved to 1.95 Å resolution. The structure has an unusual, although chymotrypsin-related, fold comprising a unique four-stranded β sheet as the N-terminal domain and a six-stranded β barrel as the C-terminal domain. A tightly bound zinc ion, essential for the stability of HRV2-2A pro , is tetrahedrally coordinated by three cysteine sulfurs and one histidine nitrogen. The active site consists of a catalytic triad formed by His18, Asp35 and Cys106. Asp35 is additionally involved in an extensive hydrogen-bonding network. Modelling studies reveal a substrate-induced fit that explains the specificity of the subsites S4, S2, S1 and S1Ј. The structure of HRV2-2A pro suggests the mechanism of the cis cleavage and its release from the polyprotein.

cDNA clones representing the entire genome of human rhinovirus 2 have been obtained and used to determine the complete nucleotide sequence. The genome consists of 7102 nucleotides and possesses a long open reading frame of 6450 nucleotides; this reading frame is initiated 611 nucleotides from the 5' end and stops 42 nucleotides from the polyA tract. The N-terminal sequences of three of the viral capsid proteins have been elucidated, thus defining the positions of three cleavage sites on the polyprotein. The extensive amino acid sequence homology with poliovirus and human rhinovirus 14 enabled the other cleavage sites to be predicted. Cleavages in the 3' half of the molecule appear to take place predominantly at Gln-Gly pairs, whereas those in the 5' half (including the capsid proteins) are more heterogeneous.

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