Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus

To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies

Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function

The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxoviruses. To explore the contribution of the cysteines in the extracellular domain to the fusion activity of HRSV F protein, each cysteine was changed to serine. Mutation of cysteines 37, 313, 322, 333, 343, 358, 367, 393, 416, and 439 abolished or greatly reduced cell surface expression suggesting these residues are critical for proper protein folding and transport to the cell surface. As expected, the fusion activity of these mutations was greatly reduced or abolished. Mutation of cysteine residues 212, 382, and 422 had little to no effect upon cell surface expression or fusion activity at 32°C, 37°C, or 39.5°C. Mutation of C37 and C69 in the F2 subunit either abolished or reduced cell surface expression by 75% respectively. None of the mutations displayed a temperature sensitive phenotype

Toll-like receptor 3 blockade in rhinovirus-induced experimental asthma exacerbations:A Randomized Controlled Study

BACKGROUND: Human rhinoviruses (HRVs) commonly precipitate asthma exacerbations. Toll-like receptor 3, an innate pattern recognition receptor, is triggered by HRV, driving inflammation that can worsen asthma. OBJECTIVE: We sought to evaluate an inhibitory mAb to Toll-like receptor 3, CNTO3157, on experimental HRV-16 inoculation in healthy subjects and asthmatic patients. METHODS: In this double-blind, multicenter, randomized, parallel-group study in North America and Europe, healthy subjects and patients with mild-to-moderate stable asthma received single or multiple doses of CNTO3157 or placebo, respectively, and were then inoculated with HRV-16 within 72 hours. All subjects were monitored for respiratory symptoms, lung function, and nasal viral load. The primary end point was maximal decrease in FEV1 during 10 days after inoculation. RESULTS: In asthmatic patients (n = 63) CNTO3157 provided no protection against FEV1 decrease (least squares mean: CNTO3157 [n = 30] = -7.08% [SE, 8.15%]; placebo [n = 25] = -5.98% [SE, 8.56%]) or symptoms after inoculation. In healthy subjects (n = 12) CNTO3157 versus placebo significantly attenuated upper (P = .03) and lower (P = .02) airway symptom scores, with area-under-the-curve increases of 9.1 (15.1) versus 34.9 (17.6) and 13.0 (18.4) versus 50.4 (25.9) for the CNTO3157 (n = 8) and placebo (n = 4) groups, respectively, after inoculation. All of the severe and 4 of the nonserious asthma exacerbations occurred while receiving CNTO3157. CONCLUSION: In summary, CNTO3157 was ineffective in attenuating the effect of HRV-16 challenge on lung function, asthma control, and symptoms in asthmatic patients but suppressed cold symptoms in healthy subjects. Other approaches, including blockade of multiple pathways or antiviral agents, need to be sought for this high unmet medical need

Francesco Ferrara, il primo degli economisti cafoscarini

The paper presents the important personality of the great Italian economist Francesco Ferrara who has been the first Director of the new School of Commerce founded in Venice in 1868. The paper is divided in two parts: the first part presents the main features of Francesco Ferrara as an economist, showing how he was clearly a supporter of a free-market oriented vision of the economic analysis and of the economic policy, not liking at all a vision of the economic analysis separated from the political implications, but definitely favouring a political economy vision. He was a sharp opponent of socialism, although admiring the logical power of Marx's thought, but not Marxian ideas. But he was also an opponent of intermediate visions leading to mediations in the field of economic policy. His rather radical positions led him to resign from the role of minister of Finance. In the second part the paper shows how Ferrara accepted the proposal of Luigi Luzzatti to be appointed as director of new School of Commerce of Ca' Foscari in summer 1868; the paper shows how the relations between Ferrara and Luzzatti were characterized by polemical moments, both because of the lines followed by Ferrara in appointing the professors of the new school and because of the openness shown by Luzzatti, and not liked at all by Ferrara, towards policies showing a favorable attitude towards social interventions. Eventually the disagreements were solved. Finally, the paper shows how Ferrara succeeded in appointing at Ca' Foscari some of the most important Italian economists of his time, such as Maffeo Pantaleoni

Mutational analysis of the role of the 68 bp block of the human polyomavirus BK enhancer in DNA replication: Activation of BKV DNA replication by transcriptional elements

The papovaviruses have been invaluable models for studying many of the basic molecular processes carried out by a mammalian cell, such as gene expression and DNA replication. Replication of the BKV double-stranded, circular DNA genome is dependent upon the interaction of the host cell DNA replication machinery and only one viral protein, large T-antigen, with sequences comprising the viral origin of replication. The regulatory region of BKV is very similar to other papovaviruses, such as simian virus 40 (SV40) and human polyomavirus JC (JCV), and contains overlapping elements comprising the viral origin of replication, early promoter, late promoter and three 68 bp direct repeats that act as a transcriptional enhancer element. The level of DNA replication from the BKV core origin is stimulated 2-20-fold by the presence of these transcriptional elements. The focus of this research has been to determine which sequences in the early promoter/enhancer region are involved in the stimulation of DNA replication of BKV-origin containing plasmids in vivo and in vitro. Two subelements of the proximal 68 bp repeat block were found to stimulate replication 2-3 fold above the level of the core alone. The first is called alpha, comprised of the first 21 bp of the first 68 bp repeat block of the transcriptional enhancer. The second subelement is referred to as beta ($\beta$) and is comprised of the remaining distal 47 bp which bind a member of the nuclear factor I (NF-1) family of proteins. When these two subelements are present together, they augment replication 2-20-fold depending upon the assay conditions. Mutation of the G-box region and the NF-1 site eliminated the ability of the 68 bp repeat block to augment replication, suggesting that these elements may function through the binding of nuclear proteins. However, a specific factor which binds to the a-element was never determined. None of several binding sites for transcription factors that were substituted for either the alpha or beta element were capable of stimulating DNA replication in the presence of BKV T-antigen. The degree to which these transcriptional elements augmented steady-state replication levels was found to vary depending upon (i) type of T-antigen (BKV or SV40) supplied in trans, (ii) method of supplying T-antigen, and (iii) cell line in which replication was assayed

Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein

Abstract Human respiratory syncytial virus (HRSV) is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus). That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP), to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.</p