fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

pH dependence of wt and mutant E1 homotrimer formation.³⁵S-labeled wt/ic, S1J, fus-1, andfus-1/ic virus preparations were treated at the indicated pH for 10 min at 37°C in the presence of liposomes, adjusted to neutral pH, and solubilized in SDS sample buffer for 3 min at 30°C. Samples were analyzed by SDS-PAGE and quantitated by phosphorimaging. Results are shown as percent E1 in the homotrimer band compared to total E1 and represent the mean for three experiments, with standard deviations between 1 and 33%.

Sensitivity of infection by wt, mutant, and revertant viruses to inhibition by NH₄Cl. BHK cells were pretreated with the indicated concentrations of NH₄Cl for 15 min at 37°C and then infected with wt/ic, S1J, fus-1,fus-1/ic, R43 revertant, or R46 revertant at 1 PFU/cell for 1.5 h in the continued presence of NH₄Cl. Following infection, the cells were treated with 2 μg of ACD per ml and 15 mM NH₄Cl for 30 min and then labeled with [³H]uridine for 3.5 h in the continued presence of ACD and 15 mM NH₄Cl. Infection was quantitated as the percent [³H]uridine incorporation compared to controls infected in the absence of NH₄Cl. Background incorporation by uninfected cells was subtracted from all points. Data represent the mean of three separate experiments for each virus, with standard deviations between 1 and 20%.

Sucrose gradient sedimentation profiles of wt andfus-1 viral spike proteins. ³⁵S-labeled wt (A and C) or fus-1 (B and C) virus was mixed with 1 mM liposomes, treated at the indicated pH for 10 min at 37°C, solubilized in 1.0 to 0.2% NP-40, and adjusted to pH 7.0. The samples were analyzed by centrifugation on 5 to 20% (wt/wt) sucrose gradients in buffer containing 0.1% NP-40. Gradients were centrifuged 22 h at 4°C in an SW41 rotor at 39,000 rpm and fractionated, and radioactivity was quantitated by scintillation counting. Fraction 1 represents the bottom of the gradient. The positions of the monomer (m), dimer (d), and E1 homotrimer (h) peaks are indicated. Recoveries ranged from 53 to 29%.

Sucrose gradient sedimentation profiles offus-1 and fus-1/ic viral spike proteins.³⁵S-labeled fus-1 or fus-1/ic virus was mixed with 1 mM liposomes and treated at the indicated pH for 10 min at 37°C. Samples were solubilized, neutralized, and analyzed by sucrose gradient sedimentation as for Fig. 4. Fraction 1 represents the bottom of the gradient. The positions of the monomer (m), dimer (d), and E1 homotrimer (h) peaks are indicated. Recoveries ranged from 32 to 63%.

pH dependence of E1* conformational changes. (A) Reactivity with MAb E1a-1. ³⁵S-labeled wt andfus-1 ectodomains were treated at the indicated pH for 10 min at 37°C in the presence of 1 mM liposomes, adjusted to neutral pH, solubilized in lysis buffer containing 1% Triton X-100, and immunoprecipitated with MAb E1a-1. E1* precipitation was quantitated by SDS-PAGE and phosphorimaging and compared to total E1* precipitated by a rabbit polyclonal antibody. Data represent the mean of three separate experiments. (B) E1* homotrimer formation.³⁵S-labeled wt SFV and fus-1 ectodomains were treated at the indicated pH for 10 min at 37°C in the presence of liposomes, adjusted to neutral pH, and solubilized in SDS sample buffer for 3 min at 30°C. E1 homotrimer formation was quantitated by SDS-PAGE and phosphorimaging. Data represent means of three separate experiments.

Amino acid sequence differences among wt, mutant, and revertant virus structural proteins. The E2 isoleucine 12 change responsible for the fus-1 phenotype is marked (∗). No amino acid sequence differences were observed between wt/ic and wt SFV or between fus-1 and fus-1/ic. The positions of the SFV structural protein coding regions are marked.