Essential role of dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation

Abstract

Dengue virus envelope protein (E) contains two N-linked glycosylation sites, at Asn-67 and Asn-153. The glycosylation site at position 153 is conserved in most flaviviruses, while the site at position 67 is thought to be unique for dengue viruses. N-linked oligosaccharide side chains on flavivirus E proteins have been associated with viral morphogenesis, infectivity, and tropism. Here, we examined the relevance of each N-linked glycan on dengue virus E protein by removing each site in the context of infectious viral particles. Dengue viruses lacking Asn-67 were able to infect mammalian cells and translate and replicate the viral genome, but production of new infectious particles was abolished. In addition, dengue viruses lacking Asn-153 in the E showed reduced infectivity. In contrast, ablation of one or both glycosylation sites yielded viruses that replicate and propagate in mosquito cells. Furthermore, we found a differential requirement of N-linked glycans for E secretion in mammalian and mosquito cells. While secretion of E lacking Asn-67 was efficient in mosquito cells, secretion of the same protein expressed in mammalian cells was dramatically impaired. Finally, we found that viruses lacking the carbohydrate at position 67 showed reduced infection of immature dendritic cells, suggesting interaction between this glycan and the lectin DC-SIGN. Overall, our data defined different roles for the two glycans present at the E protein during dengue virus infection, highlighting the involvement of distinct host functions from mammalian and mosquito cells during dengue virus propagation.

FIG. 1.

Replication of DV glycosylation mutants in mosquito cells. (A) IF assays of C6/36 cells transfected with DV RNAs, WT and mutated in the glycosylation site at Asn-67 (N67Q), Asn-153 (N153Q), and in both sites (N67Q+N153Q). The IF analysis was performed at 7 days after RNA transfection using polyclonal antibodies against DV-2. Images were taken at ×200 magnification. (B) Western blots of DV WT E and glycosylation mutants from C6/36 cell lysates at 7 days postinfection. The lysates were treated with Endo H (+) or buffer control (−). (C) IF analysis of C6/36 cells at 3 and 7 days postinfection with WT DV and the N67Q, N153Q, and N67Q+N153Q glycosylation mutants. Infection was normalized based on the amount of viral RNA in the inoculum.

FIG. 2.

Replication of DV glycosylation mutants in BHK cells. (A) IF assays of BHK cells transfected with DV WT RNA and mutants in the glycosylation site at Asn-67 (N67Q), Asn-153 (N153Q), and in both sites (N67Q+N153Q). The IF analysis was performed at 5 days after RNA transfection using polyclonal antibodies against DV-2. Images were taken at ×200 magnification. (B) IF analysis of BHK cells at 1, 2, and 3 days postinfection with WT DV and the N67Q, N153Q, and N67Q+N153Q glycosylation mutants. Infection was normalized based on the amount of viral RNA in the inoculum. (C) Production of viral particles from BHK cells infected with WT, N67Q, N153Q, and N67Q N153Q viruses was determined by quantification of viral RNA using fluorogenic RT-PCR. RNA copy numbers per ml are shown at different times postinfection. Error bars indicate standard errors of the means. (D) Secretion of E protein from cells infected with WT virus and E glycosylation mutants. The media of infected BHK cells were used for Western blot analysis. Samples of the WT and N67Q and N153Q mutant viruses collected at different times postinfection as indicated above the gel were analyzed.

FIG. 3.

Decreased infectivity of DV particles bearing the N153Q mutation in the E protein. (A) Western blots of WT DV E and the N153Q glycosylation mutant from BHK cell lysates at 3 days postinfection. The lysates were treated with PNGase F (+) or buffer control (−). (B) The top panel shows a representative image comparing the plaque phenotype of WT DV with that of the N153Q mutant virus. The plot shows one-step growth curves of WT DV and the N153Q mutant in BHK cells. The cells were infected at an MOI of 0.01, and titers were determined at each time point by plaque assay. Error bars indicate standard errors of the means. At 2 and 4 days, as indicated by the arrows, the supernatants of infected cells were used to extract and quantify the viral RNA by fluorogenic RT-PCR. Values are means ± standard errors. (C) The PFU/RNA ratios obtained in supernatants at 2 and 4 days postinfection are shown for the WT and N153Q mutant viruses. (D) Comparison of stability of WT and N153Q DV. Viral stocks were incubated at 37°C, aliquots were removed at different times, and the amounts of infectious particles were determined by plaque assay in BHK cells. Error bars indicate standard errors of the means.

FIG. 4.

Replication of DV-2 bearing the N67Q mutation in different mammalian cells. (A) BHK cells were infected with equal amounts of WT DV and N67Q DV infectious particles. Cells were harvested at 18, 24, 48, and 72 h postinfection, and viral infectivity was quantified by FACS analysis using mAb 4G2. Values are the means ± standard errors of triplicate experiments. (B) Binding of WT DV and the N67Q mutant. FACS analyses detecting cell-associated viral antigens in BHK and Vero cells are shown. The cells were incubated with medium as control (gray) or WT or N67Q DV corresponding to 50 genome equivalents of DV/cell (black), kept at 4°C, and analyzed after 2 h. The numbers in parentheses represent the means of the fluorescence intensities. The data are representative of two independent experiments. (C) Infection of BHK and Vero cells with WT DV and the N67Q mutant. The cells were infected with equivalent genome copy numbers of WT DV and the N67Q mutant (determined by quantitative PCR). The cells were stained for intracellular E protein production 24 h postinfection and analyzed by FACS. The data are representative of three independent experiments.

FIG. 5.

Secretion of DV-2 E glycosylation mutants expressed in mosquito and mammalian cells. Soluble forms of WT DV-2 E protein and glycosylation mutants were expressed using the SFV vector either in C6/36 cells or BHK cells. (A) Western blots of WT DV-2 E protein and glycosylation mutants 1 day postinfection with SFV vector in cell extracts (C) and in supernatants (S), as indicated above each gel. The extracts and supernatants of uninfected cells (UI) were used as negative controls. The E glycoproteins were detected by using mAb 1D4 directed against the C9 tag peptide introduced into the C terminus of E. Molecular markers are shown on the right. (B) Kinetics of processing and secretion of DV-2 E protein in BHK and C6/36 cells. The WT E and glycosylation mutants were pulse-labeled with [³⁵S]Cys/Met. Cytoplasmic extracts and supernatants were subjected to immunoprecipitation using mAb 1D4 at 1, 3, 5, and 7 h postinfection with SFV. Radiolabeled E proteins immunoprecipitated from cell extracts or from the medium, as indicated above each gel, are shown for each virus. Molecular markers are shown on the left. (C) Quantification of the secreted E proteins shown in panel B. The amount of WT E and the N67Q and N153Q E mutants was determined as a function of time using ImageJ software. These data are representative of two independent experiments.

FIG. 6.

Reporter DV as a tool to study viral replication. (A) Replication of reporter virus DV-R in BHK cells. The top panel shows a schematic representation of the DV-R genome. The boxes denote the coding sequences of the bicistronic construct, including the viral sequences in the first cistron and the Renilla luciferase (Ren) coding sequence in the second cistron. The bottom panel shows a one-step growth curve comparing WT DV and DV-R. BHK cells were infected at an MOI of 0.1. Viral titers were determined by plaque assays at different times. (B) Time courses of luciferase activity determined in cytoplasmic extracts of DV-R-infected cells in the presence or absence of inhibitors heparin (20 μg/ml) (Hep.) and adenosine analogue (25 μM) (RdRp Inh.). (C) Time courses of luciferase activity determined in cytoplasmic extracts of BHK cells infected with WT DV-R and the N67Q glycosylation mutant. Error bars indicate standard errors of the means.

FIG. 7.

Glycosylation at Asn-67 enhances DV infection of DC-SIGN-expressing cells. (A) Raji and Raji-DC-SIGN cells were infected with equal amounts of WT and N67Q DV infectious particles. The cells were stained for intracellular E protein production 24 h postinfection and analyzed by FACS as described above. The data are representative of three independent experiments. (B) Immature DCs were infected with WT and N67Q DV at different MOIs, stained for intracellular E antigen production, and analyzed by FACS 24 h postinfection. (C) Time courses of replication of WT DV-2 and the N67Q and N153Q mutants in Raji cells expressing DC-SIGN. The cells were infected with equivalent amounts of WT, N67Q, and N153Q DV infectious particles. Viral infection was quantified by FACS analysis at 18, 24, 48, and 72 h postinfection. Values are the means of duplicate experiments. These data are representative of three independent experiments.