Article Figures & Data
Figures
- FIG. 1.
Locations of second-site revertants of the H230A mutant in the E1*HT structure. Two of the three chains of the E1*HT (PDB entry 1RER) are represented in light gray, and the third chain is color-coded as follows: domain I in red, the first elongation of domain II in orange with the fusion loop at the tip, the second elongation of domain II in yellow with the cd loop at the tip, the linker between domains I and III in purple, domain III in blue, and the N-terminal portion of the stem present in the structure in black. Histidine 230 is represented as a red stick structure. Second-site mutations are represented by their van der Waals volumes and are color-coded as follows: the srf mutations in cyan (the srf-4 mutation on the a β-strand, the srf-5 mutation on the f β-strand, and the srf-3 mutation in the ij loop), the T70A mutation (bc loop, shown on two E1 chains) and its accompanying F200L mutation (gh loop) in green, the T234R mutation (j β-strand) in pink, the G83D mutation (fusion loop) in brown, and the three mutations found in combination at 37°C (the S120Y, Q187L, and M228I mutations, on the e and g β-strands and in the ij loop, respectively) in violet-blue. Note that the S120P and Q187L mutations were also found as single second-site mutations at 28°C (Table 1). This figure was prepared using PyMOL (6).
- FIG. 2.
Virus growth kinetics in BHK cells. BHK cells were infected with the pseudorevertants, H230A-srf3, or wt virus at a multiplicity of infection of 0.01 infectious centers (IC)/cell and incubated at 28°C (A) or 37°C (B) for the indicated times. At each time point, the culture media were collected and the progeny virus titers were determined by infectious center assay at 28°C (A) or plaque assay at 37°C (B). A representative example of two experiments is shown.
- FIG. 3.
Formation and stability of wt and mutant E1 homotrimers. 35S-labeled wt (a), H230A-srf3 mutant (b), H230A-srf4 mutant (c), and H230A-srf5 mutant (d) viruses were mixed with liposomes and treated at pH 8.0 (lanes 1 and 3) or at pH 5.5 (lanes 2, 4, and 5). The SDS resistance of the E1HT was assessed by solubilization in SDS sample buffer for 3 min at 30°C followed by SDS-PAGE (lanes 1 and 2). The trypsin resistance of the E1HT was determined by trypsin digestion (lanes 3 and 5) followed by trichloroacetic acid precipitation and detection by SDS-PAGE. Control samples (lane 4) were digested in the presence of soybean trypsin inhibitor. The positions of E1HT, E2, and E1 are indicated. A representative example of three assays of each mutant is shown.
- FIG. 4.
Cholesterol requirements of wt and mutant SFV infection. Control (black bars) and cholesterol-depleted (hatched or white bars) C6/36 mosquito cells were infected with serial dilutions of the indicated virus stocks. The cells were then incubated overnight in the presence of NH4Cl to prevent secondary infection, and infected cells were quantitated by immunofluorescence. The infectivity of each virus on control cells was normalized to 1 × 106 infectious centers (IC)/ml (27). Data shown are averages of two determinations with the range indicated. The hatched bars indicate mutants with a cholesterol dependence significantly lower than that of wt SFV, and the white bars indicate the wt SFV and those mutants with a significantly higher cholesterol dependence.
- FIG. 5.
Proposed model for the action of the second-site revertants of the E1 H230A mutant. The suggested mechanism for each group of revertants is illustrated using the surface representation of the SFV E1*HT structure with one monomer color-coded as in Fig. 1. The missing portion of the stem and the TM domain are represented by light-blue and dark-gray cylinders, respectively. The H230A mutation is shown in pink towards the tip of the yellow region of domain II. The second-site mutations are indicated in dark blue in each panel. (A) The hinge mutations act to change the angle of the hinge, as shown by the angled black arrow in which the angle position approximates the distance of the mutations from the domain II tip. Only the srf-4 mutation is visible on the external trimer surface. (B) The G83D mutation acts via charge repulsion to splay apart the domain II tips, as indicated by the short black arrows. (C) The T234R mutation acts to promote the packing of the stem within the groove of the H230A mutant trimer, as indicated by the arrow on the stem.
Tables
- TABLE 1.
Pseudorevertants of the H230A mutant
Selection temp (°C) Second-site mutationa Change in nucleotide sequenceb No. of independent isolates 28 G83D GGC → GAC 1 L44F TTG → TTT 2 V178A GTC → GCC 5 S120P TCT → CCT 1 Q187L CAG → CTG 2 T70A ACT → GCT F200L TTC → TTG 1 37 Q187L CAG → CTG S120Y TCT → TAT 1 M228I ATG → ATA T234R ACA → AGA 1 ↵ a Shown are second-site mutations present in addition to the H230A mutation in the pseudorevertants. L44F is the same amino acid change found in the srf-4 mutant, and V178A is the same amino acid change found in the srf-5 mutant.
↵ b Nucleotide changes found in the pseudorevertants are highlighted in bold.
