The Efficiency of p27 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 subtypes A and B (RSV/A and RSV/B). 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 data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell. IMPORTANCE RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained 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 contributes to the stability of the pre-F conformation.

Keywords: F protein; RSV; RSV subtypes; cleavage; p27; peptide 27; prefusion conformation; respiratory syncytial virus.

FIG 1

p27 is present in the F protein of infectious sucrose-purified RSV (spRSV) A and B. Equal amounts of spRSV were resolved by reducing SDS-PAGE gel and analyzed by Western blotting. A representative blot of duplicates is shown. Membranes were probed with palivizumab (anti-site II), RSV7.10 (anti-p27), or D25 (anti-site Ø) monoclonal antibodies. F0, uncleaved F protein (~70 kDa); F1+p27, partially cleaved p27 remained on the N terminus of F1 subunit (~60 kDa); F1, fully cleaved subunit (~50 kDa).

FIG 2

p27 and F protein in the prefusion conformation can be detected at quantifiable amounts on the surface of spRSVs. Areas under the curve (AUC) of paired measures of p27 and site II or site Ø and site II probed with monoclonal antibody palivizumab (anti-site II), RSV7.10 (anti-p27), or D25 (anti-site Ø) for spRSV/A/Bernett (A) and spRSV/B/BA (B) are shown. Site II was used as a surrogate for total F protein, and site Ø is a pre-F-specific antigenic site. Ratios of the AUC of p27 to site II and site Ø to site II determined relative proportions of p27 and pre-F to total F protein on spRSV/A/Bernett (C) and spRSV/B/BA (D). Error bars are standard deviations (n = 4 replicates). Statistical significances were determined by unpaired parametric t test in GraphPad Prism, assuming Gaussian population distribution. Correlations were calculated with a two-tailed test. P values of <0.05 were considered significant, with a 95% confidence interval. ****, P < 0.0001. ns, not significant.

FIG 3

Progressions of infection and F protein cleavage are different between HEp-2 and A549 cell lines infected with RSV/A or RSV/B. Percentage of double-positive RSV-infected HEp-2 (A and C) or A549 (B and D) cells per day postinoculation is shown. Cells were infected with RSV/A/Bernett, RSV/A/ON, RSV/A/Tracy, RSV/B/18537, or RSV/B/BA at an MOI of 0.07. Cells were costained with fluorescence-conjugated MAb (palivizumab [anti-site II], RSV7.10 [anti-p27], or D25 [anti-site Ø]). Antigenic site II is surrogate measure of total F protein, while antigenic site Ø is pre-F specific.

FIG 4

F proteins on spRSV harboring partially cleaved p27 show prefusion conformation with higher thermal stability. Changes in OD at 450 nm from indirect ELISA analysis of site II, site Ø, and p27 from spRSV/A/Bernett and spRSV/B/BA are shown. Individual spRSV aliquots were heated for 10 min at different temperatures (x axis) before ELISA (A and B). Differences in F protein content between spRSVs were adjusted by adopting site II as measure of total F protein. Changes in MAb binding to site Ø and p27 were determined at each temperature by the changes in site Ø/site II and p27/site II ratios relative to those at 25°C (C and D). Error bars are standard deviations (n = 3 replicates).

FIG 5

RSV F proteins on the surface of infected cells containing partially cleaved p27 have higher stability of the prefusion conformation. Percentages of RSV-infected cells with double-positive populations (p27+site II or site Ø+site II) after temperature-induced conformational change of F protein on the cell surface are shown. HEp-2 or A549 cells were infected with RSV/A/Bernett or RSV/B/BA (MOI = 0.07), incubated at 36°C, and harvested at 3 dpi. Aliquots of cell suspension were then heated for 10 min at increasing temperatures prior to staining and acquisition by imaging flow cytometry. (A) HEp-2 cells infected with RSV/A/Bernett; (B) HEp-2 cells infected with RSV/B/BA; (C) A549 cells infected with RSV/A/Bernett; (D) A549 cells infected with RSV/B/BA.