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The majority of human rhinoviruses use intercellular adhesion molecule 1 (ICAM-1) as a cell surface receptor. Two soluble forms of ICAM-1, one corresponding to the entire extracellular portion [tICAM(453)] and one corresponding to the two N-terminal immunoglobulin-like domains [tICAM(185)], have been produced, and their effects on virus-receptor binding, virus infectivity, and virus integrity have been examined. Results from competitive binding experiments indicate that the virus binding site is largely contained within the two N-terminal domains of ICAM-1. Virus infectivity studies indicate that tICAM(185) prevents infection by direct competition for receptor binding sites on virus, while tICAM(453) prevents infection at concentrations 10-fold lower than that needed to inhibit binding and apparently acts at the entry or uncoating steps. Neutralization by both forms of soluble ICAM-1 requires continual presence of ICAM-1 during the infection and is largely reversible. Both forms of soluble ICAM-1 can alter rhinovirus to yield subviral noninfectious particles lacking the viral subunit VP4 and the RNA genome, thus mimicking virus uncoating in vivo, although this irreversible modification of rhinovirus is not the major mechanism of virus neutralization.

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...

Mutations in the KRAS gene, especially affecting the codon 12 and 13 region of the Kras protein, have implications in the treatment of certain cancer types. Because of the current potential for the FDA to oversee laboratory developed procedures (LPDs), it is imminently important that the validation of such tests be published to ensure that it is shown that these LPDs are properly reviewed and properly tested within the CLIA guidelines, thus keeping LPDs within the CLIA regulatory domain. We have developed a pyrosequencing assay that can detect eleven mutations in the codon 12 and 13 position of the KRAS gene. Our validation consisted of a sensitivity study in which a purified mutant PCR product was introduced into a wildtype DNA background and diluted down to undetectable levels. This sensitivity test was done on six different KRAS codon 12/13 mutants to determine the detection limit of the assay. Intra- and inter-assay precision and assay accuracy was determined by comparing assay results of over 40 total samples, both mutant and wild type, over five days carried out by two technicians. The results will show that this KRAS pyrosequencing assay falls within the acceptance criteria for sensitivity, accuracy and precision. Citation Format: Matthew L. Poulin, Ann Meyer, E. Andrew Mead, Jessica Xu, Ryan Drennan, Liyin Yan. The validation of a pyrosequencing KRAS mutation detection assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1829.