Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion

Abstract

Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 64.4 A. During protein export, one of the two oligosaccharides of NS1 is processed into an endo-beta-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell.

FIG. 1

Analysis of immunoaffinity-purified soluble extracellular NS1 by SEC. (a) Immunoaffinity-purified NS1 was submitted to SEC on an S300 gel filtration column, and the single peak was concentrated by ultrafiltration and analyzed on a 4 to 20% gradient SDS-polyacrylamide gel stained by Coomassie blue. The protein was either unheated or heated to 95°C for 3 min prior to electrophoresis. (b) By comparison with protein standards used to calibrate the SEC column, the NS1 protein exhibited a Stokes radius (R_S) of 64.4 Å, corresponding to an apparent molecular mass of 300 kDa.

FIG. 2

Purified extracellular NS1 is a hexamer that can be converted to its dimeric subunit in the presence of the nonionic detergent nOG, as demonstrated by chemical cross-linking. (a) Subsequent to SEC, the protein was concentrated to 0.5 mg/ml by ultrafiltration and treated with final concentrations of 0, 0.5, 5, and 50 mM DMS. The resulting products were placed in nonreducing Laemmli sample buffer, separated on a 4 to 20% gradient acrylamide gel, and stained with Coomassie blue. One sample, treated with 50 mM DMS, was treated for 3 min at 95°C prior to electrophoresis to dissociate noncovalently linked oligomers. (b) Purified NS1 was treated overnight at 37°C with 0, 0.5, or 1% nOG and submitted or not submitted to 25 mM DMS for 1 h. Proteins were separated without heat denaturation on a 4 to 20% gradient acrylamide gel and detected by immunoblotting with MAb 3D1.4.

FIG. 3

Hexameric NS1 appears as a unique species by electron microscopy and SAXS analysis. (a) Negatively stained sample of purified NS1 at 0.2 mg/ml observed by electron microscopy at 80 kV. The image shows a field containing NS1 particles in random orientations. Dimensions are quite homogeneous, with a diameter of roughly 11 nm. Occasionally, particles lying with a twofold (large circle) or a threefold (small circle) axis of symmetry perpendicular to the plane of the figure are seen. Bar, 0.1 μm. (b) Guinier plot of purified NS1 at a protein concentration of 6.2 mg/ml. The radius of gyration (see text) calculated from the slope, R_g, is 5 ± 0.1 nm. Note that the curve is linear from very small angles (lower s values), showing that there is a single form of NS1 protein in solution. The molecular mass estimated from extrapolation to s = 0 is 300 ± 50 kDa.

FIG. 4

Analysis of the nature and accessibility of the two oligosaccharides of extracellular NS1. (a) Extracellular NS1 was mock treated (mt) or treated for 1 h at 37°C with endo H (H), endo F (F), or NPGase F (N) in its monomeric form. Samples were placed in nonreducing Laemmli sample buffer and separated by SDS–12% PAGE, and the resulting products were detected by immunoblotting after transfer to nitrocellulose. “p” indicates the presence of a polymannose-type oligosaccharide; “c” indicates a complex-type oligosaccharide on the protein. NS1(p)₀ is the deglycosylated form of the protein. (b) Purified NS1 was either mock treated or treated with NPGase F for 1 h at 37°C in 10 mM sodium phosphate buffer, pH 7.5. One sample was further treated by endo H after the pH was readjusted to 5.5 with 50 mM citrate buffer. All samples were subjected to SDS–12% PAGE with or without preliminary heat denaturation in nonreducing Laemmli sample buffer.

FIG. 5

Altered secretion of NS1 from infected cells treated with N-glycan processing inhibitors. (a) Cells were infected with Den virus at an m.o.i. of 3. At 7 h postinfection, swainsonine (Sw) and DMJ were added for 20 h at concentrations of 5 μM and 1 mM, respectively. Proteins were then radiolabeled for 3.5 h; immunoprecipitated with Den virus-specific PAb from cell lysates, total supernatants (spnt), or PEG-treated supernatants; separated by SDS-PAGE; and analyzed by autoradiography. (b) NS1-related radioactive signals from PEG-treated supernatants immunoprecipitated with the PAb were quantified directly from the gels with a PhophorImager. Average values of at least three independent experiments and corresponding standard deviations are reported on the graph. mt, mock treatment.