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. 2003 Jan;77(2):1337-46.
doi: 10.1128/jvi.77.2.1337-1346.2003.

Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR

George Lin  1 , Graham Simmons, Stefan Pöhlmann, Frédéric Baribaud, Houping Ni, George J Leslie, Beth S Haggarty, Paul Bates, Drew Weissman, James A Hoxie, Robert W Doms
Affiliations

Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR

George Lin et al. J Virol. 2003 Jan.

Abstract

The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N-glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N-glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N-glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N-glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.

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Figures

FIG. 1.
FIG. 1.
HIV-1, HIV-2, and SIV gp120 binding to CD4, chemokine receptors, or DC-SIGN(R). gp120 produced in 293T cells was bound to receptor expressing cells with or without sCD4 as indicated, and the bound protein was analyzed by SDS-PAGE and Western blot. Unbound gp120 is also shown in panels A and B (lanes labeled gp120). (A) HIV-1 gp120 proteins JR-FL and HXBc2 were bound to CD4, DC-SIGN, DC-SIGNR, or no receptor (pcDNA3). (B) HIV-2 gp120 proteins SBL/ISY and VCP were additionally evaluated on CXCR4. VCP, a CD4 independent Env, can bind directly to CXCR4, whereas SBL/ISY gp120 cannot. (C) SIVmac316 gp120 was evaluated on CCR5 or CCR5 with an aspartic acid at position 13 (CCR5/N13D), which confers more efficient binding of SIV gp120 (36).
FIG. 2.
FIG. 2.
Glycosidase analysis of receptor bound HIV-1 or HIV-2 gp120. (A) HIV-1 HXBc2 or (B) HIV-2 VCP gp120 was bound to cells expressing the indicated receptor. Cells were then washed and lysed. The gp120 in lysates was precipitated, digested with Endo H, PNGase F, or left untreated (UnTx), and then analyzed by SDS-PAGE and Western blot. Input gp120 (lanes labeled gp120) was also digested to serve as a control.
FIG. 3.
FIG. 3.
Temperature, kinetic, and differential glycosylation analysis of receptor bound HIV-2 or SIV gp120. gp120 produced in 293T cells was bound to receptor expressing cells and analyzed as described in Fig. 1. (A) VCP gp120 was bound to cells expressing CXCR4, DC-SIGN, or no receptor (pcDNA3) for 1 h either at 37 or at 4°C in the presence of 0.1% sodium azide. (B) SIVmac239 gp120 was bound to cells expressing CD4 or DC-SIGN at 37°C for 1 to 60 min as indicated. (C) SIVmac239 gp120 generated in the absence (left panel) or presence (right panel) of 1.0 mM DMJ was bound to cells expressing CD4, DC-SIGN, DC-SIGNR, or no receptor (pcDNA3) and then analyzed by SDS-PAGE and Western blot. Unbound gp120 (lanes labeled gp120) is also shown. Results with HIV-2 or SIV gp120s are shown because of the more pronounced molecular weight change with these proteins; however, results obtained with HIV-1 gp120s were similar (data not shown).
FIG. 4.
FIG. 4.
Receptor binding and glycosylation characteristics of Env from viruses grown in T-cell lines, PBMCs, pr MDMs or in the presence of DMJ. Supernatant from HIV- or SIV-infected cells was used in receptor binding assays. Bound gp120 was analyzed by SDS-PAGE and Western blot. HIV-1, HIV-2, and SIV grown in T-cell lines (A), PBMCs (B), or MDMs (C) were bound to cells expressing CD4, DC-SIGN, DC-SIGNR, or no receptor. (D and E) HIV-1 Ba-L virus from PBMCs or MDMs (D) or SupT1/CCR5 cells (E) grown in the presence or absence of 2.5 mM DMJ (absence labeled as UnTx and presence labeled as DMJ) was digested with Endo H (H) or PNGase F (F) or left untreated (UnTx) and then analyzed by SDS-PAGE and Western blot for gp120.
FIG. 5.
FIG. 5.
HIV and SIV gp120 binding to MDDCs. gp120 produced in 293T cells was bound to CD4 expressing QT6 cells or MDDCs with or without 100 μg of mannan or 20 μg of anti-CD4 MAb 19 (αCD4 MAb)/ml and then analyzed by SDS-PAGE and Western blot.
FIG. 6.
FIG. 6.
Infection by Ebola virus GP pseudovirions and analysis of GP glycosylation. (A) 293T cells mock transfected or transfected with DC-SIGN, DC-SIGNR, or ASGP-R were infected with 500 TCID50 of HIV-luciferase reporter viruses pseudotyped with VSV-G, EboZ-GP, EboS-GP, or EboZ-GP generated in the presence of 2.5 mM DMJ or MDM-derived EboZ-GP. Values are represented as the percent infection, calculated by using luciferase activity normalized to mock-transfected cells. Mean values plus the standard error of the mean are represented. (B) EboZ-GP and EboS-GP obtained from pseudovirions were incubated with the indicated lectin-biotin conjugates and then precipitated with streptavidin-agarose and analyzed by SDS-PAGE and Western blot for GP. Lectins are identified as follows: Vivia villosa lectin (VVL), Ricinus communiz agglutinin (RCA120), concanavalin A (ConA), Datura stramonium lectin (DSL), Erythrina cristagalli lectin (ECL), wheat germ agglutinin (WGA), Galanthus nivalis lectin (GNL), peanut agglutinin (PNA), Jacalin, and Ulex europaeus agglutinin (UEA). Unbound cell lysate is also shown.
FIG. 7.
FIG. 7.
Infection by MLV Env or VSV-G pseudovirions. Doxycycline-induced DC-SIGN or DC-SIGNR 293 T-Rex cells or control 293 T-Rex cells were infected with p24 normalized HIV-luciferase reporter viruses pseudotyped with MLV Env or VSV-G generated in the presence or absence of 2.5 mM DMJ. Values are represented as the percent infection, calculated by using luciferase activity normalized to control 293 T-Rex cells. Mean values plus the standard error of the mean are represented.
FIG. 8.
FIG. 8.
Location of high-mannose or complex N-glycans on the HIV Env trimer. A model of the HIV Env trimer based on optimization of quantifiable surface parameters (30) is shown in three different views, each rotated 90° about a horizontal axis. The top panel is a view from the virus. The middle panel is a side view with the viral membrane above and the target cell below. The bottom panel is a view from the target cell. The left column is an α-carbon worm trace with gp120 in brown and CD4 in yellow. The protein proximal Man3GlcNAc2 pentasaccharide core conserved between high-mannose and complex N-glycans is shown in cyan and modeled as described previously (58). The right column depicts the solvent-accessible surface of gp120 with high-mannose N-glycans in blue, complex N-glycans in black (32), and the rest of the surface in white. (This figure was kindly provided by Peter D. Kwong.)
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