TABLE 2.
Summary of properties of nine F proteins with polar or charged substitutions at critical positionsa
| Mutation(s) | Monomer | FA | CSE | TCS (°C) |
|---|---|---|---|---|
| None (wt) | – | 4+ | 100.0 ± 0.00 | 60–62 |
| L437D | A | 0 | 99.2 ± 21.2 | ND |
| L437Q | A | 0–1+ | 98.1 ± 4.6 | ND |
| L482D | B | 0 | 56.2 ± 9.4 | ND |
| L482Q | B | 0–1+ | 93.4 ± 7.0 | ND |
| L506D | B | 0 | 126.7 ± 27.5 | ND |
| L506Q | B | 0 | 114.3 ± 21.1 | ND |
| K508E | B | 0 | 89.9 ± 17.5 | 60–62 |
| K508R | B | 0 | 101.8 ± 24.6 | ND |
| L506D K508E | B/B | 0 | 61.1 ± 7.7 | 60–62 |
Mutations indicate the positions of the substitutions. The monomer is the F monomer in which the mutation occurs, defining one cavity of the trimer. Fusion activity (FA) was monitored by a transient F/H-induced cell-cell fusion assay. A fusion score was attributed to the standard and mutated F proteins as follows: 0, no fusion; 1+, limited fusion; 2+, moderate fusion; 3+, strong fusion; and 4+, massive fusion. Cell surface expression (CSE) was monitored by immunofluorescence (anti-FLAG staining) followed by flow cytometry (normalized to that of F-wt). The conformational state of F at 37°C was assessed with previously reported conformation-sensitive MAbs; in all cases, the MAb recognized prefusion F. The temperature of conformational switching (TCS) is the temperature at which F trimers switch conformation from the prefusion to the postfusion state (assessed by immunofluorescence [IF] and flow cytometry with previously described conformation-sensitive anti-F monoclonal antibodies). ND, not determined; “–,” not applicable.