Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein

Effect of β-ME on the elastase digestion of the E1 or E1* homotrimer. [³⁵S]methionine-labeled virus (A) or ectodomains (B) were low-pH treated in the presence of liposomes as for 1B and C. Samples were then incubated in 0.25% β-ME for 30 min at 37°C and digested with elastase (125 μg/ml) in 0.5% TX-100 for the indicated times at 37°C, and PMSF was added to a final concentration of 5 mM. The samples were then incubated in SDS sample buffer at 30°C for 3 min or reduced at 95°C and alkylated prior to SDS-PAGE on a 13% acrylamide gel with the Tris-glycine buffer system (A) or an 11% acrylamide gel with the Tris-Tricine buffer system (B). The labels refer to the elastase-truncated homotrimer (HTΔL), E1 fragments (E1-L1, E1-L2, and E1-L3), and E1* fragments (E1*-L1 to -L6). # (B) denotes an unidentified fragment in lanes 2 and 3 that is seen in some samples early in digestion but is not part of the stable homotrimer. * indicates higher-order oligomers of the E1 protein, and the arrowheads on the left indicate molecular mass standards as in Fig. 1C.

Trypsin digestion of E1 homotrimer (HT) formed in the absence of target membranes. [³⁵S]methionine-labeled virus was acid treated at pH 5.5 for 3 min at 20°C in the absence of liposomes. After neutralization, samples were incubated in the presence (+) or absence (−) of 0.25% β-ME for 30 min at 37°C and then digested with trypsin (125 μg/ml) in 0.5% TX-100 at 37°C as indicated. Control samples were incubated without trypsin and analyzed by SDS-PAGE as for Fig 1B. * indicates higher-order oligomers of the E1 protein.

PNGase F treatment of the E1* homotrimer. [³⁵S]methionine-labeled ectodomains were mixed with liposomes (1 mM) and treated at pH 5.5 for 10 min at 37°C to form the E1* homotrimer (HT). Samples were then incubated in the absence (−) or presence (+) of 0.25% β-ME for 30 min at 37°C, followed by treatment with PNGase F (10,000 U/ml) for 3 h at 37°C and/or trypsin (125 μg/ml) for 1 h at 37°C. Samples were reduced and analyzed as for Fig. 1B. The open arrow marks the position of deglycosylated E1* and E2*. The solid arrow marks the position of the E1*-T1 fragment.

Gradient flotation analysis of the elastase-truncated E1* homotrimer. (A) [³⁵S]methionine-labeled ectodomains were used to generate E1* homotrimers, and the preparations were digested with elastase for the indicated times, all as for Fig. 2B but in the absence of any detergent. The samples were then analyzed for their association with liposomes by flotation on sucrose step gradients (see Materials and Methods). The top (fractions 1 to 4; T) and bottom (fractions 5 to 7; B) fractions of each gradient were pooled, concentrated by acid precipitation, and then analyzed on 11% acrylamide gels after reduction and alkylation of the samples. (B) [³⁵S]methionine-labeled ectodomains were mixed with liposomes (1 mM), treated at pH 5.5 for 10 min at 37°C, and neutralized, and the E1* homotrimer was isolated by flotation on a sucrose step gradient. The top fractions of the gradient (fractions 1 to 4) were pooled and then split into three aliquots. One sample was electrophoresed without further treatment (lane 1). One sample was reanalyzed in another sucrose step gradient without any other treatment (Refloat; lanes 2 and 3). One sample was incubated in 0.25% β-ME for 30 min at 37°C, digested with elastase (250 μg/ml) for 2 h at 37°C in the absence of detergent, and then analyzed by flotation on another sucrose step gradient (Elastase; lanes 4 and 5). The gradients were fractionated, and the samples were analyzed as for panel A.