The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization are Dependent upon the Method of Virus Purification

Abstract

Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses.

Imaging Flow Cytometry and Confocal Immunofluorescence Microscopy of Virus-Host Cell Interactions

“…The relative infectivity of a virus, or the ratio of infective to defective viral particles, can vary between viruses. Infectivity is also influenced by experimental factors such as the host cell line or the method used to purify viral stocks ( Bilawchuk et al., 2017 ). It is easier to image viruses with high infectivity to particle ratios, such as respiratory syncytial virus (RSV) and poxvirus.…”

“…Unlike confocal microscopy, IFC captures an image of every cell and then quantifies the fluorescence of thousands of individual cells within a single run to generate statistically robust datasets. In virology, however, IFC is often performed in parallel with confocal microscopy to generate observations that are statistically robust and backed by 3-dimensional imaging ( Bilawchuk et al., 2017 ; Griffiths et al., 2020 ). The high-throughput and highly-quantitative nature of IFC has been helpful to characterize subtle host-pathogen interactions and limit the analysis of imaging artifacts ( Figure 2 ).…”

“…Using GFP-tagged syndecan constructs and immunolabeled SARS-CoV-2 viral proteins, data gathered by IFC revealed the colocalization and increased cellular uptake of the S1 subunit of the SARS-CoV-2 spike protein with syndecan-4 ( Hudak et al., 2021 ). In another example, IFC was used to image ‘patching’ of the RSV coreceptor NCL at the cell surface ( Bilawchuk et al., 2017 ; Griffiths et al., 2020 ). Shown in Figure 1C , the colocalization of fluorescent NCL constructs and RSV-GFP particles within lipid rafts on the cell surface were quantified, demonstrating focal colocalization that increased following infection ( Bilawchuk et al., 2017 ; Griffiths et al., 2020 ).…”

“…In another example, IFC was used to image ‘patching’ of the RSV coreceptor NCL at the cell surface ( Bilawchuk et al., 2017 ; Griffiths et al., 2020 ). Shown in Figure 1C , the colocalization of fluorescent NCL constructs and RSV-GFP particles within lipid rafts on the cell surface were quantified, demonstrating focal colocalization that increased following infection ( Bilawchuk et al., 2017 ; Griffiths et al., 2020 ). IFC is a powerful tool to elucidate novel virus entry receptors.…”

Immune-Modulation by the Human Respiratory Syncytial Virus: Focus on Dendritic Cells

“…Covering the nucleocapsid, yet beneath the envelope is the matrix protein M, which has been reported to travel to the nucleus of infected cells during the replication cycle of hRSV to inhibit the transcription of host genes and was recently described to interact with actin within infected cells, likely contributing to the transport of virion components into budding virions (30, 32, 33). Importantly, the virion envelope is covered on its surface by the attachment glycoprotein G, which may be dispensable for infection in some cells (34–36), the fusion F glycoprotein which is a type-I integral membrane protein that binds nucleolin for cell infection (37, 38), and the transmembrane protein SH, which forms a viroporin that transports cationic ions (39, 40). Importantly, to date, there is accumulating data that describes the molecular interactions between hRSV structural components, which has allowed establishing an overall comprehensive scenario of how the virus' components are coordinately assembled within infected cells to favor its replication and exit (30).…”