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

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.     

West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection

The C-type lectins DC-SIGN and DC-SIGNR bind mannose-rich glycans with high affinity. In vitro, cells expressing these attachment factors efficiently capture, and are infected by, a diverse array of appropriately glycosylated pathogens, including dengue virus. In this study, we investigated whether these lectins could enhance cellular infection by West Nile virus (WNV), a mosquito-borne flavivirus related to dengue virus. We discovered that DC-SIGNR promoted WNV infection much more efficiently than did DC-SIGN, particularly when the virus was grown in human cell types. The presence of a single N-linked glycosylation site on either the prM or E glycoprotein of WNV was sufficient to allow DC-SIGNR-mediated infection, demonstrating that uncleaved prM protein present on a flavivirus virion can influence viral tropism under certain circumstances. Preferential utilization of DC-SIGNR was a specific property conferred by the WNV envelope glycoproteins. Chimeras between DC-SIGN and DC-SIGNR demonstrated that the ability of DC-SIGNR to promote WNV infection maps to its carbohydrate recognition domain. WNV virions and subviral particles bound to DC-SIGNR with much greater affinity than DC-SIGN. We believe this is the first report of a pathogen interacting more efficiently with DC-SIGNR than with DC-SIGN. Our results should lead to the discovery of new mechanisms by which these well-studied lectins discriminate among ligands.     

N-linked glycosylation of west nile virus envelope proteins influences particle assembly and infectivity

West Nile virus (WNV) encodes two envelope proteins, premembrane (prM) and envelope (E). While the prM protein of all WNV strains contains a single N-linked glycosylation site, not all strains contain an N-linked site in the E protein. The presence of N-linked glycosylation on flavivirus E proteins has been linked to virus production, pH sensitivity, and neuroinvasiveness. Therefore, we examined the impact of prM and E glycosylation on WNV assembly and infectivity. Similar to other flaviviruses, expression of WNV prM and E resulted in the release of subviral particles (SVPs). Removing the prM glycosylation site in a lineage I or II strain decreased SVP release, as did removal of the glycosylation site in a lineage I E protein. Addition of the E protein glycosylation site in a lineage II strain that lacked this site increased SVP production. Similar results were obtained in the context of either reporter virus particles (RVPs) or infectious lineage II WNV. RVPs or virions bearing combinations of glycosylated and nonglycosylated forms of prM and E could infect mammalian, avian, and mosquito cells (BHK-21, QT6, and C6/36, respectively). Those particles lacking glycosylation on the E protein were modestly more infectious per genome copy on BHK-21 and QT6 cells, while this absence greatly enhanced the infection of C6/36 cells. Thus, glycosylation of WNV prM and E proteins can affect the efficiency of virus release and infection in a manner that is cell type and perhaps species dependent. This suggests a multifaceted role for envelope N-linked glycosylation in WNV biology and tropism.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.     

DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.     

Interaction of HIV-1 with dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-expressing cells is influenced by gp120 envelope modifications associated with disease progression

Dendritic cells can enhance the replication of HIV-1 in CD4(+) lymphocytes through the interaction of the gp120 envelope protein with such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin. The variable loops of gp120 have previously been shown to modulate the interaction of HIV-1 with its principal receptor CD4 and its various coreceptors, namely CCR5 and CXCR4. Here, we utilized a panel of molecular cloned viruses to identify whether gp120 modifications can influence the virus interaction with immature dendritic cells or a cell line expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (Raji-DC-SIGN). The viruses encompass the R5, R5X4 and X4 phenotypes, and are based upon V1V2 and V3 sequences from a patient with disease progression. We found that dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin enhancement of virus replication can be modulated by the V1V2 length, the overall V3 charge and N-linked glycosylation patterns; similar results were observed with immature dendritic cells. Viruses with higher V3 charges are more readily transferred to CD4(+) lymphocytes when the V1V2 region is longer and contains an additional N-linked glycosylation site, whereas transfer of viruses with lower V3 charges is greater when the V1V2 region is shorter. Viruses differing in the V1V2 and V3 regions also demonstrated differential capture by Raji-DC-SIGN cells in the presence of mannan. These results indicate that the interaction between HIV-1 and immature dendritic cells via such molecules as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin may have a role in selecting viruses undergoing transmission and evolution during disease progression.     

Cryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGN

Dengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 A resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.     

Structure of immature West Nile virus

The structure of immature West Nile virus particles, propagated in the presence of ammonium chloride to block virus maturation in the low-pH environment of the trans-Golgi network, was determined by cryo-electron microscopy (cryo-EM). The structure of these particles was similar to that of immature West Nile virus particles found as a minor component of mature virus samples (naturally occurring immature particles [NOIPs]). The structures of mature infectious flaviviruses are radically different from those of the immature particles. The similarity of the ammonium chloride-treated particles and NOIPs suggests either that the NOIPs have not undergone any conformational change during maturation or that the conformational change is reversible. Comparison with the cryo-EM reconstruction of immature dengue virus established the locations of the N-linked glycosylation sites of these viruses, verifying the interpretation of the reconstructions of the immature flaviviruses.     

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.     

HIV-1 N-glycan composition governs a balance between dendritic cell-mediated viral transmission and antigen presentation

The natural function of dendritic cells (DCs) is to capture and degrade pathogens for Ag presentation. However, HIV-1 can evade viral degradation by DCs and hijack DCs for migration to susceptible CD4(+) T lymphocytes. It is unknown what factors decide whether a virus is degraded or transmitted to T cells. The interaction of DCs with HIV-1 involves C-type lectin receptors, such as DC-specific ICAM-3-grabbing nonintegrin, which bind to the envelope glycoprotein complex (Env), which is decorated heavily with N-linked glycans. We hypothesized that the saccharide composition of the Env N-glycans is involved in avoiding viral degradation and Ag presentation, as well as preserving infectious virus for the transmission to target cells. Therefore, we studied the fate of normally glycosylated virus versus oligomannose-enriched virus in DCs. Changing the heterogeneous N-linked glycan composition of Env to uniform oligomannose N-glycans increased the affinity of HIV-1 for DC-specific ICAM-3-grabbing nonintegrin and enhanced the capture of HIV-1 by immature DCs; however, it decreased the subsequent transmission to target cells. Oligomannose-enriched HIV-1 was directed more efficiently into the endocytic pathway, resulting in enhanced viral degradation and reduced virus transfer to target cells. Furthermore, Env containing exclusively oligomannose N-glycans was presented to Env-specific CD4(+) T cells more efficiently. Taken together, our results showed that the HIV-1 N-glycan composition plays a crucial role in the balance between DC-mediated Ag degradation and presentation and DC-mediated virus transmission to target cells. This finding may have implications for the early events in HIV-1 transmission and the induction of antiviral immune responses.     

The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specific glycans on the E2 envelope protein

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.     

CD40 ligand enhances dengue viral infection of dendritic cells: a possible mechanism for T cell-mediated immunopathology

We have previously shown that dengue virus (DV) productively infects immature human dendritic cells (DCs) through binding to cell surface DC-specific ICAM-3-grabbing nonintegrin molecules. Infected DCs are apoptotic, refractory to TNF-alpha stimulation, inhibited from undergoing maturation, and unable to stimulate T cells. In this study, we show that maturation of infected DCs could be restored by a strong stimulus, CD40L. Addition of CD40L significantly reduced apoptosis of DCs, promoted IL-12 production, and greatly elevated the IFN-gamma response of T cells, but yet did not restore T cell proliferation in MLR. Increased viral infection of DCs was also observed; however, increased infection did not appear to be mediated by DC-specific ICAM-3-grabbing nonintegrin, but rather was regulated by decreased production of IFN-alpha and decreased apoptotic death of infected DCs. Because CD40L is highly expressed on activated memory (but not naive) T cells, the observation that CD40L signaling results in enhanced DV infection of DC suggests a possible T cell-dependent mechanism for the immune-mediated enhancement of disease severity associated with some secondary dengue infections.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Role of N-linked glycans of HCV glycoprotein E1 in folding of structural proteins and formation of viral particles

Envelope proteins E1 and E2 of the hepatitis C virus (HCV) play a major role in the life cycle of a virus. These proteins are the main components of the virion and are involved in virus assembly. Envelope proteins are modified by N-linked glycosylation, which is supposed to play a role in their stability, in the assembly of the functional glycoprotein heterodimer, in protein folding, and in viral entry. The effects of N-linked glycosylation of HCV protein E1 on the assembly of structural proteins were studied using site-directed mutagenesis in a model system of Sf9 insect cells producing three viral structural proteins with the formation of virus-like particles due to the baculovirus expression system. The removal of individual N-glycosylation sites in HCV protein E1 did not affect the efficiency of its expression in insect Sf9 cells. The electrophoretic mobility of E1 increased with a decreasing number of N-glycosylation sites. The destruction of E1 glycosylation sites N1 or N5 influenced the assembly of the noncovalent E1E2 glycoprotein heterodimer, which is the prototype of the natural complex within the HCV virion. It was also shown that the lack of glycans at E1 sites N1 and N5 significantly reduced the efficiency of E1 expression in mammalian HEK293 T cells.     

Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1 is engaged both in viral attachment and viral immune evasion mechanisms in the infected host. Besides several N-linked glycans, gC-1 contains numerous O-linked glycans, mainly localized in two pronase-resistant clusters in the N-terminal domain of gC-1. In the present study we construct and characterize one gC-1 mutant virus, in which two basic amino acids (114K and 117R) in a putative O-glycosylation sequon were changed to alanine. We found that this modification did not modify the N-linked glycosylation but increased the content of O-linked glycans considerably. Analysis of the O-glycosylation capacity of wild-type and mutant gC-1 was performed by in vitro glycosylation assays with synthetic peptides derived from the mutant region predicted to present new O-glycosylation sites. Thus the mutant peptide region served as a better substrate for polypeptide GalNAc-transferase 2 than the wild-type peptide, resulting in increased rate and number of O-glycan attachment sites. The predicted increase in O-linked glycosylation resulted in two modifications of the biological properties of mutant virus-that is, an impaired binding to cells expressing chondroitin sulfate but not heparan sulfate on the cell surface and a significantly reduced plaque size in cultured cells. The results suggested that basic amino acids present within O-glycosylation signals may down-regulate the amount of O-linked glycans attached to a protein and that substitution of such amino acid residues may have functional consequences for a viral glycoprotein involving virus attachment to permissive cells as well as viral cell-to-cell spread.     

Routes of inoculation and the immune response to a resolving genital flavivirus infection in a novel murine model

The prolonged, abnormal immune response patterns produced by many sexually transmitted viruses have been intensively studied. Because normal antiviral immune responses in the vagina are less well-defined, we developed a resolving murine model using vaginal inoculation with the flavivirus, West Nile virus. Infection resulted in 12% mortality, with sterile protective immunity to vaginal or systemic re-challenge. B-cell numbers increased in the vaginal mucosa from day 1-7 after primary infection, while similar increases in B220(+), CD4(+) and CD8(+) lymphocytes in the draining lymph node were delayed by 48 h. By day 4 postinfection, a MHC-II(+) dendritic cell population became depleted from the stroma and formed aggregates below the basement membrane at points of demonstrable epithelial infection. In contrast, primary systemic or intradermal inoculation resulted in 80-90% mortality, but also conferred protective sterile immunity to vaginal West Nile virus re-challenge. Intravaginal and intradermal immunization elicited comparable, accelerated accumulation of larger B-cell numbers in the mucosa and draining lymph node upon intravaginal re-challenge than systemic immunization. However, accumulation of CD4(+) T cells in both sites in the intradermally immunized group was significantly greater than in intravaginally or systemically immunized mice. Accelerated accumulation of dendritic cells occurred at periodic sub-basement membrane sites in the absence of detectable virus 1 day after vaginal re-challenge, irrespective of the route of immunization. These data illustrate the diversity of possible effective immune responses to West Nile virus in the vaginal mucosa. They show primary vaginal inoculation produces effective immunity to flavivirus infection with lower mortality than other routes and suggest a local role for vaginal mucosal dendritic cells in both primary and secondary responses.     

Immunochemical and single molecule force spectroscopy studies of specific interaction between the laminin binding protein and the West Nile virus surface glycoprotein E domain II

ELISA and Western blot immunochemical data attest an effective and highly specific interaction of the surface glycoprotein E domain II (DII) of the tick born encephalitis and Dengue viruses with the laminin binding protein (LBP). Based on a highly conservative structure of the DII in different flaviviruses we propose a similarly effective interaction between the LBP and the DII of the surface glycoprotein E of the West Nile virus. We report the results of studies of this interaction by immunochemical and single molecule force spectroscopy methods. The specific binding between these species is confirmed by both methods.     

Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections

Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.