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R J Colonno

Publications and source records attributed to R J Colonno.

At least 55 records · Page 3Linked to original sources
PubMed1990

Bacterial expression of antibody fragments that block human rhinovirus infection of cultured cells.

Human rhinoviruses are the major causative agents of the common cold in humans and have been divided into major and minor groups based on receptor specificity. cDNAs encoding the light and heavy chains of a murine monoclonal antibody that recognizes the major group receptor were cloned, abundantly expressed in Escherichia coli, and renatured into Fab fragments that blocked virus binding and protected HeLa cell monolayers from rhinovirus infection. Elimination of the cysteines normally bridging the heavy and light chains yielded molecules indistinguishable from wild-type Fab fragments in virus binding assays. Single-chain antibodies with covalently linked light and heavy variable domains were also expressed and showed receptor binding and cell protection activities. These recombinant antibody fragments are potentially useful in preventing or treating common colds in humans.

Amino Acid Sequence↗
PubMed1990

Antibodies that block rhinovirus attachment map to domain 1 of the major group receptor.

The vast majority of human rhinovirus serotypes utilize the intercellular adhesion molecule 1 (ICAM-1) as the attachment site on susceptible cells. Twelve murine monoclonal antibodies were isolated and shown by competition binding studies to recognize three distinct, nonoverlapping epitopes on the ICAM-1 receptor. Titration of three antibodies representing each of the binding sites demonstrated that they were equally effective at blocking viral attachment. By using in vitro transcription and translation systems, a series of progressive C-terminal truncations of ICAM-1 molecules was generated. Immunoprecipitation of these fragments with each of the three antibodies indicated that all three epitopes reside within the first 82 amino acids of the receptor. Attempts to demonstrate specific binding of these in vitro-synthesized receptor fragments to virions were unsuccessful. The inability to show virion binding was most likely due to a failure of the lysates to properly glycosylate the receptor molecule, since native, unglycosylated receptor molecules isolated from cell membranes were also inactive in virus binding assays.

Animals↗
PubMed1989

Biochemical characterization of a glycoprotein required for rhinovirus attachment.

Human rhinoviruses attach to specific receptors located on the surfaces of host cells as a first step in viral infection. A 90-kDa cell surface protein was previously shown to be involved in the attachment of human rhinoviruses to susceptible cells (Tomassini, J. E., and Colonno, R.J. (1986) J. Virol. 58, 290-295). Digestion of purified receptor protein with various glycosidases revealed that 30% of its molecular mass was comprised of complex-type oligosaccharides, one-third being contributed by sialic acid. The presence of sialic acid was confirmed by demonstrating that wheat germ lectin can inhibit the attachment of rhinoviruses to host cell membranes, while lectins of other sugar specificities had no effect. The oligosaccharides were shown to be N-linked by tunicamycin treatment of host cells and by N-glycanase digestion. Seven N-linked glycosylation sites were detected by partial digestion of the receptor oligosaccharides with N-glycanase. Native receptor protein had an isoelectric focusing point of 4.2, compared to 5.3 for the deglycosylated protein. Studies of virus and antibody binding to neuraminidase-treated host cell membranes suggested that although carbohydrates may be involved in host-virus interaction, the receptor carbohydrate is not the predominant component of the cellular receptor site.

Attachment Sites, Microbiological↗
PubMed1989

cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1.

A 90-kDa surface glycoprotein was previously isolated and shown to be required for infection by the "major" group of human rhinovirus (HRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the major group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed.

Amino Acid Sequence↗
PubMed1989

Effect of 2'-nor-cyclic GMP against guinea pig cytomegalovirus infection.

Cyclic phosphate derivative of DHPG, 2'-nor-cGMP [9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]-guani ne phosphate-oxide] was evaluated for activity against guinea pig cytomegalovirus (GPCMV) infection in cultured guinea pig embryo cells and in guinea pigs. By virus yield reduction and plaque reduction assays, 2'-nor-cGMP was demonstrated to be 15- to 20-fold more potent against GPCMV infection than its parental drug DHPG. The selectivity index of 2-nor-cGMP was 110, which was 10-fold higher than that of DHPG. In cultured cells, 2'-nor-cGMP attained maximal antiviral activity when added to the cells within 12 h postinfection. In the studies on GPCMV infection in guinea pigs, 2'-nor-cGMP administered subcutaneously once daily (5 mg/kg per day) for 8 days, starting 24 after virus inoculation, significantly suppressed GPCMV infectivity titers in the blood, spleen, lung, and salivary gland during acute infection (10 days postinfection) as compared with sham-treated infected animals. A greater reduction of GPCMV infectivity titers in the salivary gland was noted during chronic infection (i.e., 24 days postinfection). Clinically, splenomegaly and peripheral lymphocytosis were significantly modified as compared with the sham-treated animals (P less than 0.05). The drug, administered at this dosage, was reasonably tolerated by the guinea pigs and showed clinical benefit.

Animals↗
PubMed1989

Inhibition of rhinovirus attachment by neutralizing monoclonal antibodies and their Fab fragments.

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pI 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pIs of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13- to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.

Antibodies, Monoclonal↗
PubMed1989

Cleavage of small peptides in vitro by human rhinovirus 14 3C protease expressed in Escherichia coli.

The 3C region of human rhinovirus 14 was expressed in Escherichia coli. The microbially synthesized protease was functional, since the expressed precursor underwent autoproteolytic processing to generate mature molecules of the expected molecular weight and antigenicity. Mutation of the putative active-site Cys-146 residue to an alanine resulted in the synthesis of unprocessed precursor molecules. Large quantities of the 20-kilodalton protease were purified by a simple purification protocol, and the resulting molecule was shown to be biologically active in vitro against synthetic peptides corresponding to the 2C-3A cleavage site. This site was cleaved with high efficiency and fidelity and was used to generate kinetic data on the 3C protease. The protease exhibited sensitivity to Zn2+, was capable of cleaving five of seven rhinovirus cleavage site peptides tested with variable efficiency, and could distinguish authentic substrate peptides from control peptides containing the dipeptide cleavage sequence pair Gln-Gly.

3C Viral Proteases↗
PubMed1989

Chimeric picornavirus polyproteins demonstrate a common 3C proteinase substrate specificity.

Cross-species proteolytic processing was demonstrated by the 3C proteinases of human rhinovirus 14 and coxsackievirus B3 on poliovirus-specific polypeptide precursors. Chimeric picornavirus cDNA genomes were constructed in a T7 transcription vector in which the poliovirus 3C coding region was substituted with the corresponding allele from human rhinovirus 14 or coxsackievirus B3. In vitro translation and processing of the polypeptides encoded by the chimeric genomes demonstrated that the proteolytic processing of poliovirus P2 region (nonstructural) proteins could be functionally substituted by the heterologous proteinases. In contrast, the 3C proteinase activities expressed from the chimeric genomes were incapable of recognizing the poliovirus-specific processing sites within the capsid precursor. Since the amino acid sequences flanking and inclusive of the P2 region cleavage sites of the three viruses are not stringently conserved, these results provide evidence for the existence of common conformational determinants necessary for 3C-mediated processing.

Chimera↗
PubMed1988

Modification of experimental rhinovirus colds by receptor blockade.

Human rhinovirus (HRV) infection can be inhibited in vitro by antibody directed against the cellular receptor for the major HRV group representing 90% of serotypes. We assessed the prophylactic effectiveness and safety of intranasally administered rhinovirus receptor murine monoclonal antibody (RRMA) in two double-blind, place-controlled, randomized studies of volunteers experimentally inoculated with HRV-39. In the first study, RRMA administration (135 micrograms/subject in 9 applications, -17 to +48 h) did not reduce infection (RRMA 12/15 vs. placebo 13/15) or illness (8/12 vs. 7/13) rates or modify the clinical course of experimental HRV-39 colds. In the second trial, a higher RRMA dosage (1 mg/subject in 10 applications, -3 to +36 h), similarly did not reduce overall infection (11/13 vs. 12/13) or illness (7/11 vs. 9/12) rates, but was associated with a 1-2 day delay in the onset of viral shedding and cold symptoms and with significant reductions in viral titers and nasal symptoms on the second day after challenge and in mucus weights on the third day after challenge. No toxicity related to RRMA was recognized. The results indicate that intranasal RRMA modified infection and illness after experimental HRV-39 challenge and suggest that blockade of host cell receptors offers a novel antiviral approach against HRV infections.

Administration, Intranasal↗
PubMed1988

Evidence for the direct involvement of the rhinovirus canyon in receptor binding.

Evidence is presented that indicates a deep crevice located on the surface of human rhinovirus type 14 is involved in virion attachment to cellular receptors. By using mutagenesis of an infectious cDNA clone, 11 mutants were created by single amino acid substitutions or insertions at positions 103, 155, 220, 223, and 273 of the structural protein VP1. Seven of the recovered mutants had a small plaque phenotype and exhibited binding affinities significantly lower than wild-type virus. One mutant, in which glycine replaced proline at amino acid position 155, showed a greatly enhanced binding affinity. Single-cycle growth kinetics suggested that 5 of the mutants had delayed growth cycles due to intracellular deficiencies apart from receptor binding.

HeLa Cells↗
PubMed1988

Characterization of human rhinoviruses displaced by an anti-receptor monoclonal antibody.

The attachment of rhinoviruses to cellular receptors was studied by displacing bound virus particles with an anti-receptor monoclonal antibody. The two serotypes studied differed significantly with respect to the temperature dependence of displacement and the nature of the particles displaced. Binding was shown to be a two-step process, the first of which is reversible and is seen when viruses are bound either to isolated cell membranes or to cells at lower than physiological temperatures. Second-stage binding was seen with serotype 14 when bound to intact cells. Viral particles released from such cells by incubation at 37 degrees C or by anti-receptor antibody exhibited altered physical changes in the capsid and a loss of infectivity. In contrast, serotype 67 bound efficiently to cells at 37 degrees C and did not elute spontaneously but could be displaced by anti-receptor antibody to produce complete, infectious particles. Rhinoviruses labeled with [3H]myristic acid or with [35S]methionine were displaced similarly from cells or membranes by anti-receptor antibody, indicating that the majority of VP4 of rhinoviruses does not enter or remain attached to cells during either the first or second stage of virus binding. These data support the conclusion that the myristic acid moiety of VP4 is not involved in the initial viral interaction with cellular receptors.

Antibodies, Monoclonal↗
PubMed1987

A collaborative report: rhinoviruses--extension of the numbering system from 89 to 100.

To define the number of rhinovirus serotypes, cross neutralization tests and characterization studies were completed on 25 candidate prototype rhinoviruses submitted to a third phase of a collaborative program. Based on the results, 11 distinct prototype strains were designated and the numbering system was extended to include 100 rhinoviruses. In addition, recent evidence indicates that over 90% of rhinoviruses isolated in three areas of the country could be typed with antisera for rhinovirus types 1-89.

Neutralization Tests↗
PubMed1986

Use of monoclonal antibodies to identify four neutralization immunogens on a common cold picornavirus, human rhinovirus 14.

A collection of 35 mouse monoclonal antibodies, raised against human rhinovirus 14 (HRV-14), was used to isolate 62 neutralization-resistant mutants. When cross-tested against the antibodies in a neutralization assay, the mutants fell into four antigenic groups, here called neutralization immunogens: NIm-IA, -IB, -II, and -III. Sequencing the mutant RNA in segments corresponding to serotype-variable regions revealed that the amino acid substitutions segregated into clusters, which correlated exactly with the immunogenic groups (NIm-IA mutants at VP1 amino acid residue 91 or 95; NIm-II mutants at VP2 residue 158, 159, 161, or 162; NIm-III mutants at VP3 residue 72, 75, or 78; and NIm-IB mutants at two sites, either VP1 residue 83 or 85, or residue 138 or 139). Examination of the three-dimensional structure of the virus (M. G. Rossmann, E. Arnold, J. W. Erickson, E. A. Frankenberger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kamer, M. Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend, Nature [London], 317:145-153, 1985) revealed that each of the substitution clusters formed a protrusion from the virus surface, and the side chains of the substituted amino acids pointed outward. Moreover, four of the amino acid substitutions, which initially appeared to be anomalous because they were encoded well outside the cluster groups, could be traced to surface positions immediately adjacent to the appropriate viral protrusions. We conclude that three of the four antigens, NIm-IB, -II, and -III, are discontinuous. Thus, the amino acid substitutions in all 62 mutants fell within the proposed immunogenic sites; there was no evidence for alteration of any antigenic site by a distal mutation.

Antibodies, Monoclonal↗
PubMed1986

Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses.

Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage.

Animals↗
PubMed1986

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

Animals↗
PubMed1986

Isolation of a receptor protein involved in attachment of human rhinoviruses.

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.

Cell Division↗
PubMed1985

Antigenic conservation and divergence between the viral-specific proteins of poliovirus type 1 and various picornaviruses.

Immuneprecipitation analyses of various picornavirus-infected cell lysates were performed using antisera to poliovirus type 1-specific structural and nonstructural proteins. The results showed differing patterns of antigenic conservation and divergence. However, the VP3 and 2C polypeptides were strongly antigenically conserved among the large majority of these viruses. This conservation was especially notable given the degree of divergence exhibited by the other viral proteins and may be due to environmental pressure exerted by interaction with the host cell. The results, furthermore, allowed for an analysis of the evolutionary relationship of the tested viruses. This analysis showed a particularly strong antigenic relationship between the proteins of the poliovirus group and coxsackievirus A21 as well as a weaker, but significant, relationship with coxsackieviruses B1 and B3.

Antigens, Viral↗