Intercellular adhesion molecule 1 (ICAM-1) is the cellular receptor for the major group of human rhinoviruses (HRVs) and the adhesion ligand of lymphocyte function-associated antigen 1. Analysis of a series of chimeric exchanges between human and murine ICAM-1 shows that two distinct epitopes recognized by monoclonal antibodies that block rhinovirus attachment and cell adhesion map to the N-terminal first domain of ICAM-1. Furthermore the specificity for HRV binding is entirely contained within the first 88 amino acids. Mutagenesis of the four sites of N-linked glycosylation within the second domain shows that carbohydrate is not involved in virus recognition. Homologue replacement mutagenesis localizes the epitopes for virus-blocking antibodies to two regions of domain 1 predicted to form .3 strand D and the loop between the F and G strands of an immunoglobulinfold structure. Analysis of virus binding to the mutants predicts a large surface of contact between HRV and ICAM-1 domain 1 but shows that the regions most important for virus binding are coincident with the monoclonal antibody epitopes.There are more than 100 distinct serotypes of human rhinoviruses (HRVs), the primary causative agent of the common cold (1). Viral entry mediated by binding to a cellular receptor is a critical first step in the infection process and is an important determinant of viral tropism. Ninety percent of HRV serotypes utilize a common cellular receptor to initiate infection (2, 3), which we have recently shown is intercellular adhesion molecule 1, or ICAM-1 (4). ICAM-1 is a member of the IgG supergene family; it interacts with the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) (5, 6). The molecule is an integral membrane glycoprotein with an extracellular region of453 amino acids containing five domains with sequence similarity to the IgG constant regions.The three-dimensional structure of HRV14, which binds to ICAM-1, and of HRV1A, which binds to the as yet unidentified minor receptor, has been determined (7,8). Determination of the structure of HRV14 led Rossmann to propose the canyon hypothesis, which suggests that a 20-A-wide surface depression, which encircles the fivefold axis of symmetry of each icosahedral face of the virus, may contain the receptor binding site. Support for the canyon hypothesis comes from site-directed mutagenesis of canyon residues, which alter the receptor binding properties of HRV14 (9), and from studies with capsid binding drugs, which induce a conformational change in the floor of the canyon and prevent receptor binding (10). The dimensions of the canyon are sufficient to accommodate a single unpaired IgG domain, and it has recently been shown by electron microscopy that ICAM-1 and the related neural cell adhesion molecule (NCAM) have long elongated structures consistent with an end-to-end arrangement of unpaired IgG domains (11,12). The N-terminal domain of ICAM-1 is therefore likely to project furthest from the cell surface and be most accessible to virus. Furthermore...
RNA is involved in many biological functions, ranging from information storage and transfer to the catalysis of reactions involving both nucleic acids and proteins. Previous crystallographic studies on RNA oligomeric chains provide only averaged structures or information limited in resolution. The oligomer [U(U-A)6A]2 was chosen for the study of protein-RNA interactions in viruses. Its size and base composition mimic portions of the genomic RNA in alfalfa mosaic virus that bind to the amino terminus of the viral subunit. The actual sequence was designed to guarantee the formation of a single species of duplex and to facilitate the production of the pure oligomer in large quantities. The molecular structure, derived from the 2.25 A resolution X-ray diffraction data, allows the most detailed analysis of an A-RNA helix reported to date. Two kinks are observed that divide the duplex into three blocks, each close to a canonical A-helical conformation. A few intermolecular hydrogen bonds involving 2'-hydroxyl groups stabilize this peculiar conformation of the RNA, which may be related to the temperature used for the crystallization (35 degrees C). The structure demonstrates both the plasticity of the RNA molecule and the role of the 2'-hydroxyl groups in intermolecular interactions.
Both polarities ofthe satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain.Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The .aation of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.Several RNA molecules undergo autolytic cleavage to generate a 2',3'-cyclic phosphodiester and a 5'-hydroxyl as the new chain end groups. Most of these self-cleaving RNA sequences are derived from small satellite RNAs associated with plant viruses of the nepovirus and sobemovirus groups (1, 2), and most conform to a consensus secondary structure. termed a "hammerhead structure," which also applies to a self-cleaving viroid RNA (2, 3). The form ofthe satellite RNA of tobacco ringspot that is most abundantly encapsidated has a self-cleaving sequence that conforms to the hammerhead structure, whereas the self-cleaving complement of this RNA, designated sTobRV(-)RNA, does not have the conserved sequences of the consensus secondary structure.
Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17-hydroxysteroid dehydrogenases (17-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17-HSD I was detectable only in MG63 cells, albeit at low levels, while 17-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17-HSD III was absent from each bone cell type.
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