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Structure and Assembly

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

Don L. Gibbons, Margaret Kielian
Don L. Gibbons
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
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Margaret Kielian
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • For correspondence: kielian@aecom.yu.edu
DOI: 10.1128/JVI.76.3.1194-1205.2002
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  • FIG. 1.
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    FIG. 1.

    Effect of β-ME on the trypsin digestion of the E1 or E1* homotrimer. (A) Linear diagram of domains I (solid), II (hatched), and III (stippled) within the primary sequence of SFV E1 plus the putative stem region (shaded) and transmembrane domain (checkered). (Data are from reference 32). (B) [35S]methionine-labeled virus was mixed with liposomes (0.8 mM), treated at pH 5.5 for 3 min at 20°C to form the E1 homotrimer, and adjusted to neutral pH. Samples were then incubated in the indicated concentrations of β-ME for 30 min at 37°C, followed by digestion with (+) trypsin (125 μg/ml) in 0.5% TX-100 for 1 h at 37°C. The digestion was stopped by addition of PMSF to a final concentration of 5 mM, and the samples were incubated in SDS sample buffer for 3 min at 30 or 95°C as indicated and analyzed by SDS-PAGE on an 11% acrylamide gel using the Tris-glycine buffer system. The positions of the E1 homotrimer (HT), E2, E1, capsid (C), and trypsin-cleaved E1 (E1-T1) are indicated. Higher-order oligomers of the E1 protein are indicated by the asterisk. (C) [35S]methionine-labeled ectodomains were mixed with liposomes (1 mM), treated at pH 5.5 for 10 min at 37°C to form the E1* homotrimer, and adjusted to neutral pH. Samples were then incubated in 0.25% β-ME for 30 min at 37°C, digested with trypsin as for panel B for the indicated times (0 to 2.5 h), and analyzed by SDS-PAGE as for panel B, using a 10% polyacrylamide gel with the Tris-Tricine buffer system. The E1* and E2* ectodomains comigrate under these conditions. The positions of the E1* truncated homotrimer (HTΔT) and the T1, T2, and T3 fragments are indicated, and the arrowheads on the left denote the electrophoretic positions of molecular mass standards in kilodaltons.

  • FIG. 2.
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    FIG. 2.

    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.

  • FIG. 3.
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    FIG. 3.

    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.

  • FIG. 4.
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    FIG. 4.

    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.

  • FIG. 5.
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    FIG. 5.

    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.

  • FIG. 6.
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    FIG. 6.

    Gel filtration analysis of the elastase-truncated E1* homotrimer. (A) [35S]methionine-labeled ectodomains were mixed with [3H]-labeled liposomes (1 mM) and treated at pH 5.5 for 10 min at 37°C to form the E1* homotrimer. Samples were then incubated in 0.25% β-ME for 30 min at 37°C and digested with elastase (250 μg/ml) for 3 h at 37°C in the absence of detergent. PMSF was added to a final concentration of 5 mM, and the sample was chromatographed on a Superdex 200 column. The elution of radioactivity representing [35S]-labeled protein and [3H]-labeled liposomes was determined by liquid scintillation counting of a 50-μl aliquot of each of the fractions collected. The elution volumes of 3H-labeled liposomes alone or the protein standard albumin (67 kDa), aldolase (158 kDa), or catalase (232 kDa) (positions marked by long arrows) were determined by separate column runs. The short arrows on the abscissa mark the void (determined by blue dextran elution) and total elution volumes of the column. (B) The fractions of the three radioactive peaks shown in panel A were pooled, acid precipitated, and analyzed by SDS-PAGE with a Tris-glycine buffer system after reduction and alkylation of the samples (lanes 3 to 5). Lane 2 contains a sample of the digest mixture prior to chromatography and represents ∼7% of the sample loaded onto the column. Lane 1 shows a control of ectodomains treated at low pH without digestion to indicate the positions of E1*, E2*, and the homotrimer (HT).

Tables

  • Figures
  • TABLE 1.

    Analysis of E1 ectodomains by MALDI-TOF mass spectrometry

    TABLE 1.
  • TABLE 2.

    Trypsin fragments of E1 or E1* homotrimer

    Fragment nameN-terminal sequenceApparent mass (kDa)Calculated peptide mass (kDa)
    E1-T180VYTGVYPFM3838.5
    E1*-T1 80VYTGVYPF3533.8
    E1*-T2NDa 22ND
    E1*-T3YEHST66–7.9
    • ↵ a ND, not determined.

  • TABLE 3.

    Elastase fragments of E1 or E1* homotrimer

    Fragment nameN-terminal sequence(s)Apparent mass (kDa)Calculated peptide mass (kDa)
    E1-L179KVYTGVYPF3838.6
    95FCDSENTQL36.8
    97DSENTQLSE36.6
    E1-L2 102QLSEAYV3536.0
    E1-L3NDa 32ND
    E1*L1 81YTGVY3533.7
    95FCDSE32.1
    97DSENT31.8
    E1*-L2 102QLSEA3331.3
    E1*-L4 230HVPYT18.517.2
    E1*-L5 102QLSEA18.514.1
    E1*-L6ND6ND
    • ↵ a ND, not determined.

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Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein
Don L. Gibbons, Margaret Kielian
Journal of Virology Feb 2002, 76 (3) 1194-1205; DOI: 10.1128/JVI.76.3.1194-1205.2002

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Molecular Dissection of the Semliki Forest Virus Homotrimer Reveals Two Functionally Distinct Regions of the Fusion Protein
Don L. Gibbons, Margaret Kielian
Journal of Virology Feb 2002, 76 (3) 1194-1205; DOI: 10.1128/JVI.76.3.1194-1205.2002
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KEYWORDS

Membrane Glycoproteins
Semliki Forest virus
Viral Envelope Proteins
Viral Fusion Proteins

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