The efficiency of p27 peptide cleavage during in vitro respiratory syncytial virus (RSV) infection is cell line and RSV subtype dependent

Abstract

Respiratory Syncytial Virus (RSV) Fusion protein (F) is highly conserved between RSV/A and RSV/B subtypes. To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27 amino acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous immunological and cell-surface expression data show that p27 is detected on RSV F, but questions remain on how p27 effects the conformation of mature RSV F. Monoclonal antibodies against p27, Site O (pre-Fusion specific), and Site II were used to monitor RSV F conformation by ELISA and Imaging Flow Cytometry. Pre-F to post-F conformational change was induced by temperature-stress test. We found that p27 cleavage efficiency was lower on sucrose purified (sp) RSV/A than on spRSV/B. In addition, in vitro cleavage of RSV F was cell-line dependent, higher levels of p27 expression were observed on surface of RSV infected HEp-2 cells than A549 cells. Higher levels of p27 were also found on RSV/A infected cells compared to RSV/B. We observed that RSV/A F with higher levels of p27 could better sustain the pre-F conformation during the temperature-stress challenge in both spRSV and as well RSV-infected cell lines. Our findings suggest that despite F sequence similarity, the p27 of RSV subtypes is cleaved with different efficiencies, which were also dependent on the cell lines used for infection. We therefore speculate that partially cleaved p27 may confer higher stability to the pre-F and provides a fitness advantage. IMPORTANCEThe RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F protein undergoes proteolytic cleavages by furin protease resulting in the release of a 27 amino acid peptide (p27) to become fully functional. During this process, the F protein also undergoes a conformational change from metastable pre-F to highly stable post-F. For decades, the consensus in the RSV field was that p27 was not present in the fully mature RSV F protein. Therefore, the role of the p27 peptide in viral entry and the function of the partially cleaved F protein containing p27 has been overlooked. However, recent developments have shown that p27 elicits an immune response during natural infection and that p27 can be found in vitro and in animal models infected with the prototypical RSV/A strain. In this study, we were able to detect p27 on purified RSV virions and on the surface of virus-infected cells of two widely used cell lines, HEp-2 and A549 cells for both prototypical and contemporary circulating RSV strains of both subtypes. Also, higher levels of partially cleaved F protein containing p27 could better sustain the pre-F conformation during the temperature-stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 in partially cleaved F protein likely contributes to the stability of the pre-F conformation.