Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum

Abstract

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.

FIG. 1

Production of structural proteins of DEN-1 virus. At the top is a schematic representation of the orientation in the ER membranes of C, prM, and E (5, 6, 26). Signal peptides are shown as cylinders, and stop-transfer sequences are shown as rectangular blocks. The proteolytic cleavage sites are indicated by arrows (open arrow, signal peptidase cleavage site; dark arrow, furin cleavage site) (5, 6, 26, 27). The circled CHOs are potential sites for N-glycosylation (9, 17). At the bottom is shown the amino acid sequence of residues 95 to 114 of C of FGA/89, numbered from the N terminus of C (9). Below the sequence, the open arrow indicates the putative cleavage site that reacts with the NS2B/NS3 protease complex (2, 19, 28, 33, 35). Amino acids 101 to 114 are thought to constitute a transmembrane domain (; P. Desprès, V. Deubel, and F. Pénin, unpublished results). The model is not to scale.

FIG. 2

DEN virus production by glucosidase inhibitor-treated Neuro 2a cells. Neuro 2a cells were infected with strain FGA/89 of DEN-1 virus at a multiplicity of infection of 400 (7). (a) Three sets of infected cell monolayers were incubated with various doses of CST or DNJ for 6 h beginning at 19 h postinfection. Virus yield 25 h postinfection was determined by focus immunoassay titration (9). Virus titer is expressed as the virus titer in CST- or DNJ-treated cells as a percentage of that in mock-treated cells. Data were compared between groups with the Fisher and Yates t test. P < 0.05 was considered significant. n.s., not significant. (b) Viral protein synthesis in glucosidase inhibitor-treated cells. Infected Neuro 2a cells were incubated without (Control) or with 500 μM CST (+CST) or DNJ (+DNJ) for 3 h beginning 19 h postinfection. Proteins from infected cells were labeled for 1 h at 21 h postinfection. DEN virus structural proteins from cell lysate samples were immunoprecipitated with anti-prM MAb 14E9 or 15H5 or with anti-E MAb 8C2, 9D12, or 4D2 (7, 9). Samples were analyzed by SDS-PAGE under nonreducing conditions.

FIG. 3

Kinetics of FGA/89 envelope glycoprotein processing in Neuro 2a cells. Neuro 2a cells infected with DEN-1 virus (FGA/89) or mock-infected cells (No virus) were pulse-labeled for 10 min at 20 h postinfection and chased for various times. (a) Cell lysates were immunoprecipitated with anti-prM (15H5) or anti-E (4D2) MAb. (b) Samples on gels were analyzed with a PhosphorImager (Molecular Dynamics). Quantitation of the prM and E coimmunoprecipitated by MAb 15H5 (top) and the prM+E/prM ratio were used to define the kinetics of association of the prM and E proteins (bottom). Quantitation of the E protein precipitated by MAb 4D2 is shown at the bottom.

FIG. 4

Processing of FGA/89 envelope glycoproteins in Neuro 2 cells. (a) Cell lysates chased for 30 min were immunoprecipitated with MAb 15H5. Labeled antigens were eluted from complexes in SDS and β-mercaptoethanol by boiling, and the denatured samples were divided into three aliquots for endo H (H), PNGase F (F), or mock (−) digestion as previously described (36). Glycosylated (g) and nonglycosylated (ng) forms of prM and E are indicated. (b) Pulse-labeled prME complexes chased for 90 min were subjected to sedimentation analysis on sucrose gradients. Samples were immunoprecipitated from gradient fractions with MAb 15H5 and separated by SDS-PAGE. Protein E that coprecipitated with prM was quantitated with a PhosphorImager (black boxes). The sedimentation of detergent-solubilized glycoprotein E homodimer from DEN-1 virions was determined by treating highly purified virus with 1% Triton X-100 in TNI buffer at 20°C for 1 h (1). Denatured virions were run in the parallel sucrose gradient, and protein E homodimer was detected by enzyme-linked immunosorbent assay using an anti-E specific antibody (open boxes). O.D., optical density.

FIG. 5

Processing of prM and E in FGA/89-infected Neuro 2a cells treated with α-glucosidase inhibitor. Infected cells were pulse-labeled 20 h postinfection and chased in the presence of the α-glucosidase inhibitor (500 μM) or mock treated (Control). (a) Samples were immunoprecipitated with anti-DEN virus antibody. Proteins prM and E are indicated. (b and c) prM and E coimmunoprecipitated by MAb 15H5 are quantified as described in the legend to Fig. 3.

FIG. 6

Kinetics of recombinant DEN virus envelope glycoprotein processing in N2aprM+E cells. N2aprM+E cells were briefly pulse-labeled 20 h after addition of ponasterone A (5 μM) and chased for various times. (a) Cell lysates were immunoprecipitated with anti-DEN virus antibody; (b) samples on gels were quantified as described in the legend to Fig. 3.

FIG. 7

Processing of recombinant prM and E in N2aprM+E cells treated with α-glucosidase inhibitor. (a) Mock-treated (−) or CST-treated (+) cells were pulse-labeled and chased as described in the legend to Fig. 5. Cell lysates were immunoprecipitated with anti-DEN virus antibody. (b) Mock-treated (−CST) or CST-treated (+CST) samples chased for 30 min were immunoprecipitated with MAb 15H5. Immunoprecipitated proteins were directly analyzed (Control) or incubated in the presence (PGNase F) or absence (Untreated) of PGNase F as described in the legend to Fig. 4. (c) α-Glucosidase inhibitor-containing samples (CST or DNJ) or mock-treated samples (Mock) were immunoprecipitated with MAb 15H5.