Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

Abstract

All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCEWorldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein are less infectious for primary airway epithelial cells, the natural RSV target. In the study described here we identified the protease responsible, located the cleavage site, and demonstrated that cleavage likely occurs during endocytic recycling. Moreover, we showed that the infectivity of Vero cell-derived virus for primary airway epithelial cells is increased 5-fold if the virus contains a mutation in the G protein that prevents cleavage. The blocking of cleavage should improve RSV vaccine yield, consequently reducing production costs. Posttranslational cleavage of the fusion glycoprotein of many viruses plays an essential role in activation; however, cleavage of the RSV G protein is a novel example of a detrimental effect of cleavage on virus infectivity. In adults and teenagers, most respiratory syncytial virus (RSV) infections produce upper respiratory tract infection and symptoms. However, in infants, the immunocompromised, and the elderly, RSV has the potential to cause life-threatening lower respiratory tract infection (1-4). Worldwide, in 2010 ...

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

“…The presence of two bands of sG is consistent with previous observations from hRSV G (73,133,386,387). It has been hypothesised that the different species arise from dimer formation, differential glycosylation or significant cleavage of hRSV G (73,133,386,387).…”

“…Further downstream to the HDB two residues have been implicated as a cleavage site for cathepsin L between residues K209 and T210 (386). In the present study T210 and T211 were also The present study provides a template for future analyses of glycopeptides derived from hRSV G particularly highlighting the benefits of EThcD and in some instances HCD, combined with multiple enzymatic digestions.…”

“…It has been hypothesised that the different species arise from dimer formation, differential glycosylation or significant cleavage of hRSV G (73,133,386,387). Treatment of sG with Sialidase-S and Sialidase-A decreased the migration of both the 100 and 50 kDa bands, an effect that has been observed previously for hRSV G (373).…”

“…Construction of sG is described in (386), for the purposes of this work the proteins were expressed in HEK293F cells and purified using wheat germ agglutinin. …”

Respiratory syncytial virus (RSV) entry is inhibited by serine protease inhibitor AEBSF when present during an early stage of infection

“…This enzymatic processing is associated with structural changes and is required to activate the fusogenicity of the F protein [15,28]. The RSV G protein was recently shown to be subjected to cleavage by Cathepsin L during endocytic recycling in Vero cells, resulting in a smaller size, but this feature seems to occur only in Vero cells [29].…”