Purification of respiratory syncytial virus F and G proteins.

Respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract infections of infants in industrial nations. In addition, the participation of RSV in the genesis of asthma is under discussion. The RSV glycoproteins F and G have key positions in the viral pathogenesis. At present no satisfactory protein purification protocols are available for these proteins. The methods published for the G protein using preparative SDS-PAGE or immunoaffinity chromatography yield only small amounts of purified G protein that has partially lost its antigenicity. We describe a three-step purification protocol for these glycoproteins. RSV-infected HEp-2 cells were lysed by a Triton X-100 containing buffer. The viral proteins were captured by QAE-Sephadex A-50 material in a batch procedure. A first elution with 100 mM NaCl led to a crude F protein fraction, and a second elution with 300 mM NaCl led to a crude G protein fraction. The F protein was further purified on a Lentil-lectin Sepharose 4B column and finally polished using a Resource Isopropyl column. Lentil-lectin Sepharose 4B was also used to purify the G protein from the crude fraction, but polishing of the G protein was carried out on a Resource Q column. Homogenous RSV-F and RSV-G proteins were obtained by this protein purification protocol. No loss of antigenicity could be observed during this procedure as the highly purified viral proteins remain detectable by a set of monoclonal antibodies and specific antisera. The G protein was isolated as a 90000 monomer, whereas the purified F protein was recovered as a functional homodimer of 140000.

Persistence of airway hyperresponsiveness and viral antigen following respiratory syncytial virus bronchiolitis in young guinea-pigs.

Respiratory syncytial virus (RSV) bronchiolitis in infancy is known to be followed by chronic respiratory symptoms and airway hyperresponsiveness in a subgroup of patients. To further investigate the pathogenesis of RSV-induced chronic airway pathology, we infected young guinea-pigs at 4 weeks of age with RSV applied as an aerosol (n=30), and control guinea-pigs with virus-free culture medium (n=24). Infection was confirmed by positive antibody titre to RSV after 6 weeks, and by typical pathological changes of bronchiolitis after 1 week in six animals from each group. Airway hyperresponsiveness was measured weekly for 5 weeks by histamine challenge, using body-plethysmographic measurement of compressed air (CA). The provocative concentration of histamine producing significant airway obstruction (i.e. CA = 0.1 mL) (PC0.1 mL CA in mg x mL(-1)) was calculated from dose-response curves. Six weeks postinfection, the lungs were investigated for the presence of inflammation and of viral antigen by immunofluorescence and immunohistochemistry using a rabbit hyperimmune serum and monoclonal antibodies. Airway responsiveness was increased in the RSV group 1 week postinfection compared to the control group (PC0.1 mL CA median 2.50 vs >10 mg x mL(-1); p<0.001) and this persisted up to 5 weeks postinfection (PC0.1 mL CA median 1.61 vs >10 mg x mL(-1); p<0.001). During the same period, viral antigen persisted in the lungs of infected animals, although there was less inflammation at 6 weeks postinfection than at 1 week postinfection. In guinea-pigs, respiratory syncytial virus infection of the airways causes persistent airway hyperresponsiveness over a period of at least 5 weeks. During this time, viral antigen, but not inflammation, remains detectable in the lungs and might be responsible for ongoing airway hyperresponsiveness.

Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells.

In this study we could demonstrate that heparin (ED50 = 0.32 +/- 0.12 microgram/ml), but not heparan sulphate or chondroitin sulphate C is able to inhibit in vitro infection of cells by respiratory syncytial virus (RSV). In addition, this protective effect of heparin could only be observed, when heparin was present at the time of inoculation. Enzymatic digestion of cell surface glycosaminoglycans with heparinase and heparitinase, but not chondroitin sulphate ABC lyase reduced the effectiveness of RSV-infection. Affinity chromatography experiments, using immobilised heparin further demonstrated that RSV attachment protein G was able to bind specifically to heparin. Therefore heparin-like proteoglycans showed properties required for attachment of RSV to host cells.

IL-8 release from human neutrophils by the respiratory syncytial virus is independent of viral replication.

Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5-10 after 18-24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 microg/5 X 10(5) PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 microg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.

Respiratory syncytial virus G-protein modulates cytokine release from human peripheral blood mononuclear cells.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children and adults. In vivo the host has to cope with intact replicative virus, with non-replicative virus, and/or with viral structural proteins including the outer membrane G-protein. We analyzed the role of purified RSV G-protein with regard to its modulatory efficacy for interleukin (IL) -10, IL-12, and tumor necrosis factor-alpha (TNF-alpha) release from human peripheral blood mononuclear cells (PBMC). These cytokines seem to contribute to the deleterious effect in viral infections. Treatment of PBMC with RSV at a multiplicity of infection of 10 down to 0.001 induced the release of TNF-alpha, IL-10, and IL-12; also time kinetics and dose-responses differed markedly. Stimulation of PBMC with purified RSV G-protein (from 0.001 up to 10 microgram/10(6) PBMC) led only to a pronounced increase in IL-10 within a concentration range from 0.01 up to 0.5 microgram/10(6) PBMC with a maximum between 12 and 18 h of incubation. AT later time points (24, 48, and 72 h) G-protein concentrations above 1 microgram/10(6) PBMC suppressed IL-10, TNF-alpha, and IL-12 release from human PBMC. Coating of PBMC with RSV G-protein suppressed IL-10, TNF-alpha, and IL-12 release after subsequent stimulation with RSV. Our data indicate a regulatory role of RSV G-protein immune responses toward viral infection.