Biochemical analysis of the secreted and virion glycoproteins of Ebola virus

Abstract

The glycoproteins expressed by a Zaire species of Ebola virus were analyzed for cleavage, oligomerization, and other structural properties to better define their functions. The 50- to 70-kDa secreted and 150-kDa virion/structural glycoproteins (SGP and GP, respectively), which share the 295 N-terminal residues, are cleaved near the N terminus by signalase. A second cleavage event, occurring in GP at a multibasic site (RRTRR downward arrow) that is likely mediated by furin, results in two glycoproteins (GP1 and GP2) linked by disulfide bonding. This furin cleavage site is present in the same position in the GPs of all Ebola viruses (R[R/K]X[R/K]R downward arrow), and one is predicted for Marburg viruses (R[R/K]KR downward arrow), although in a different location. Based on the results of cross-linking studies, we were able to determine that Ebola virion peplomers are composed of trimers of GP1-GP2 heterodimers and that aspects of their structure are similar to those of retroviruses, paramyxoviruses, and influenza viruses. We also determined that SGP is secreted from infected cells almost exclusively in the form of a homodimer that is joined by disulfide bonding.

FIG. 1

Effects of nonionic detergent treatment on a purified virion preparation of EBO virus. Shown are autoradiograms of [³⁵S]cysteine-labeled virion proteins run on 6% (A) and 10% (B) acrylamide gels (lanes contain the same samples). Lanes 1 to 3 contain reduced samples of untreated purified EBO virions, pelleted nucleocapsids derived from detergent (TX100) treatment of the same preparation, and membrane-associated proteins (supernatant fluid from the TX100 treatment), respectively; lanes 4 to 6 contain the same samples and in the same order, except that 2-mercaptoethanol was omitted from treatment (nonreduced). Identified in the left margin are the migration positions for the structural proteins; L (polymerase) protein is putative. The question mark in panel B identifies an unknown membrane-associated protein. Asterisks identify glycoprotein bands absent from the nucleocapsid pellet and present in the supernatant fluid.

FIG. 2

Diagrammatic representation of SGP and GP molecules of EBO virus (Zaire species isolated in 1976) showing important structural features. The white N-terminal regions of SGP and GP correspond to identical (shared) sequences, while the black C termini identify sequences unique to GP or SGP molecules. The common signalase cleavage sites for both SGP and GP and the furin cleavage site for GP0 (uncleaved form of GP) (↓) were determined by N-terminal sequencing. Also shown are cysteine residues (S), predicted N-linked glycosylation sites (Y-shaped projections), a predicted fusion peptide, a heptad repeat sequence, and a transmembrane anchor sequence. In EBO viruses, the positions of these structures are conserved and their sequences are very similar or, in the case of N-linked glycosylation sites, are at least concentrated in the central region of GP.

FIG. 3

Reactivities of polyclonal antibodies to GP and SGP in Western blot and RIP assays. (A) Paired lanes of untreated (−) and endoglycosidase F/N-glycosidase F-digested (+) virion proteins labeled with [³⁵S]cysteine, run on 10% gels, and blotted onto nitrocellulose membranes. Blots were directly exposed to X-ray film (³⁵S) to identify locations of structural proteins and reacted in enzyme immunoassays (chemiluminescent) with guinea pig antisera. Arrows indicate the decreased size of virion GP1 and GP2 glycoproteins due to removal of N-linked glycans. Sera were derived from animals immunized with naked plasmid DNA which directed the expression of GP (α-GP), SGP (α-SGP), or vector-only (α-VEC) products. Asterisks identify SGP and deglycosylated SGP detected by α-SGP. Shown in panel B are lanes containing [³⁵S]cysteine-labeled proteins from an EBO virion preparation (marker) and RIP products immunoprecipitated from supernatant fluids depleted of virions (pelleted through 20% sucrose cushion) with the same antisera as used for panel A.

FIG. 4

Autoradiograms of multimeric forms of GP and SGP separated by SDS-PAGE. Numbers next to bands indicate monomeric, dimeric, and trimeric forms of GP or monomeric and dimeric forms of SGP. (A) EBO and MBG virion preparations that were untreated (lane 1), cross-linked (lane 2), and TX100 disrupted and then cross-linked (lane 3). Virion glycoproteins were labeled with [³H]glucosamine and separated on a 3.5% gel. Identified on the left are the migration positions and sizes (in kilodaltons) of cross-linked phosphorylase b marker proteins (195 kDa = dimer, 292 kDa = trimer, etc.; note that in 3.5% gels, proteins under 200 kDa migrate anomalously). (B) [³H]glucosamine-labeled EBO virion GP1 (lanes 1 and 3) and SGP immunoprecipitated from TCF (lanes 2 and 4) separated on a 3.5% gel. Samples were run under reducing (lanes 1 and 2) or nonreducing (lanes 3 and 4) conditions. The GP1 band in lane 3 corresponds to the monomeric form seen in lane 1 of panel A. GP and SGP bands in panels A and B were also verified by Coomassie blue staining of gels prior to autoradiography to identify the locations of purified virion proteins. (C) Ten percent gel containing immunoprecipitated [³⁵S]cysteine-labeled SGPs that were either untreated or cleaved with formic acid. Lanes 1 and 3 contain untreated samples of SGP; lanes 2 and 4 contain SGP digested with formic acid (Asp-Pro cleavage; asterisks identify cleavage products). Samples in lanes 1 and 2 were run under reducing conditions, while those in lanes 3 and 4 were nonreduced.

FIG. 5

Biochemical analysis of SGP and GP expressed separately through recombinant DNA techniques. Shown are the results of Western blot assays (chemiluminescent) detecting proteins separated on SDS–4 to 15% gradient polyacrylamide gels. SGP was derived from cell cultures transfected with plasmid DNA containing an unedited ORF sequence, and GP was derived from partially concentrated GP-pseudotyped retrovirus particles (GP expressed from an edited ORF sequence). Preparations were cross-linked as for Fig. 3 and analyzed under reducing (R) and nonreducing (NR) conditions. Migration positions of size markers are shown on the right; monomeric and multimeric forms are indicated on the left.

FIG. 6

Production of EBO virus SGP in pulse-chase and tunicamycin treatment experiments. (A) RIP products obtained from the TCFs of EBO-infected (+) and uninfected (−) E6 cells. Following a 10-min pulse ([³⁵S]cysteine) and chasing with cold medium, samples were taken from 20 to 240 min after the moment the radiolabel was added. (B) RIP products from the TCFs of cultures treated with tunicamycin at 1.0, 0.5, 0.25, 0.125, 0.062, 0.031, or 0.015 μg/ml (lanes 1 to 7, respectively) or untreated (lane 8).

FIG. 7

Diagrammatic representation of the structural GP. Shown is the predicted orientation of the GP1-GP2 heterodimer linked by undetermined disulfide bonding (indicated by the question mark). Intramolecular disulfide bonds that are shown come from prior predictions based on similarities to retrovirus glycoprotein structures (6). See Fig. 2 for other features of the amino acid sequence.