Fedde Groot

Publications of Fedde Groot

• Architecture and regulation of the HIV-1 assembly and holding compartment in macrophages.

  Authors: Sonja Welsch, Fedde Groot, Hans-Georg Kräusslich, Oliver T Keppler, Quentin J Sattentau

  Journal of virology. 05/2011; 85(15):7922-7.

  Productive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little isProductive infection of macrophages is central to HIV-1 pathogenesis. Newly formed virions bud into a tubular membranous compartment that is contiguous with the plasma membrane. However, little is known about the structure of this compartment and its potential regulation by infection. Here we characterized this compartment in macrophages using electron tomography and electron microscopy with stereology. We found an intricate, interconnected membrane network that constitutes a preexisting physiologic structure in macrophages but which expands in size upon HIV-1 infection. Membranes required for this expansion were apparently derived from preexisting pools of plasma membrane. Physical connections between this compartment and the extracellular milieu were frequently made by tube-like structures of insufficient diameter for virion passage. We conclude that HIV-1 induces the expansion of a complex membranous labyrinth in macrophages in which the virus buds and can be retained, with potential consequences for transmission and immune evasion.

• Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses.

  Authors: Fedde Groot, Sonja Welsch, Quentin J Sattentau

  Blood. 06/2008; 111(9):4660-3.

  Macrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4(+) T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidlyMacrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4(+) T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4(+) T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1-infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.

• The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion.

  Authors: Rogier W Sanders, Eelco van Anken, Alexei A Nabatov, I. Marije Liscaljet, Ilja Bontjer, Dirk Eggink, Mark Melchers, Els Busser, Martijn M. Dankers, Fedde Groot, Ineke Braakman, Ben Berkhout, William A Paxton

  Retrovirology. 02/2008; 5:10.

  BACKGROUND: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in variousBACKGROUND: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for approximately 50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. RESULTS: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385-418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. CONCLUSION: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.

• The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

  Authors: Rogier Sanders, van Anken Eelco, Alexei Nabatov, Liscaljet I Marije, Ilja Bontjer, Dirk Eggink, Mark Melchers, Els Busser, Martijn Dankers, Fedde Groot, Ineke Braakman, Ben Berkhout, William Paxton

  Retrovirology. 01/2008;

  Abstract Background The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in variousAbstract Background The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. Results The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. Conclusion The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.

• Sialoadhesin (CD169) expression in CD14+ cells is upregulated early after HIV-1 infection and increases during disease progression.

  Authors: Antoinette C van der Kuyl, Remco van den Burg, Fokla Zorgdrager, Fedde Groot, Ben Berkhout, Marion Cornelissen

  PLoS ONE. 02/2007; 2(2):e257.

  BACKGROUND: Sialoadhesin (CD169, siglec-1 or Sn) is an activation marker seen on macrophages in chronic inflammatory diseases and in tumours, and on subsets of tissue macrophages. CD169 is highlyBACKGROUND: Sialoadhesin (CD169, siglec-1 or Sn) is an activation marker seen on macrophages in chronic inflammatory diseases and in tumours, and on subsets of tissue macrophages. CD169 is highly expressed by macrophages present in AIDS-related Kaposi's sarcoma lesions. It is also increased on blood monocytes of HIV-1 infected patients with a high viral load despite antiretroviral treatment. METHODOLOGY/PRINCIPAL FINDINGS: We investigated expression of sialoadhesin in untreated HIV-1 and HHV-8 infected patients, by real-time PCR and FACS analysis to establish its expression in relation to infection and disease progression. Patients analysed were either HIV-1 seroconverters (n = 7), in the chronic phase of HIV-1 infection (n = 21), or in the AIDS stage (n = 58). Controls were HHV-8 infected, but otherwise healthy individuals (n = 20), and uninfected men having sex with men (n = 24). Sialoadhesin mRNA was significantly elevated after HIV-1, but not HHV-8 infection, and a further increase was seen in AIDS patients. Samples obtained around HIV-1 seroconversion indicated that sialoadhesin levels go up early in infection. FACS analysis of PBMCs showed that sialoadhesin protein was expressed at high levels by approximately 90% of CD14(+) and CD14(+)CD16(+)cells of HIV-1(+) patients with a concomitant 10-fold increase in sialoadhesin protein/cell compared with uninfected controls. CONCLUSIONS/SIGNIFICANCE: We have shown that sialoadhesin is induced to high levels on CD14(+) cells early after HIV-1 infection in vivo. The phenotype of the cells is maintained during disease progression, suggesting that it could serve as a marker for infection and probably contributes to the severe dysregulation of the immune system seen in AIDS.

• Stimulated plasmacytoid dendritic cells impair human T-cell development.

  Authors: Heike Schmidlin, Wendy Dontje, Fedde Groot, Suzanne J Ligthart, Arnaud D Colantonio, Monique E Oud, Esther J Schilder-Tol, Marcel Spaargaren, Hergen Spits, Christel H Uittenbogaart, Bianca Blom

  Blood. 01/2007; 108(12):3792-800.

  Thymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into theThymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into the thymus, allowing for interactions between thymic pDCs and precursor cells. We demonstrate that in vitro-generated pDCs stimulated with CpG or virus impaired the development of human autologous CD34(+)CD1a(-) thymic progenitor cells into the T-cell lineage. Rescue by addition of neutralizing type I interferon (IFN) antibodies strongly implies that endogenously produced IFN-alpha/beta is responsible for this inhibitory effect. Consistent with this notion, we show that exogenously added IFN-alpha had a similar impact on IL-7- and Notch ligand-induced development of thymic CD34(+)CD1a(-) progenitor cells into T cells, because induction of CD1a, CD4, CD8, and TCR/CD3 surface expression and rearrangements of TCRbeta V-DJ gene segments were severely impaired. In addition, IL-7-induced proliferation but not survival of the developing thymic progenitor cells was strongly inhibited by IFN-alpha. It is evident from our data that IFN-alpha inhibits the IL-7R signal transduction pathway, although this could not be attributed to interference with either IL-7R proximal (STAT5, Akt/PKB, Erk1/2) or distal (p27(kip1), pRb) events.

• Opposing roles of blood myeloid and plasmacytoid dendritic cells in HIV-1 infection of T cells: transmission facilitation versus replication inhibition.

  Authors: Fedde Groot, Toni M M van Capel, Martien L Kapsenberg, Ben Berkhout, Esther C de Jong

  Blood. 10/2006; 108(6):1957-64.

  CD11c(+) myeloid dendritic cells (MDCs) and CD11c(-) CD123(+) plasmacytoid DCs (PDCs) have been identified as main human DC subsets. MDCs are professional antigen-presenting cells for T cells, andCD11c(+) myeloid dendritic cells (MDCs) and CD11c(-) CD123(+) plasmacytoid DCs (PDCs) have been identified as main human DC subsets. MDCs are professional antigen-presenting cells for T cells, and include Langerhans cells, dermal DCs, and interstitial DCs. They have been associated with HIV-1 capture and sexual transmission, whereas PDCs play an important role in the innate immune responses to different types of viruses, including HIV-1. To compare the influence of MDCs and PDCs on HIV-1 infection of T cells, we isolated donor-matched MDCs and PDCs from peripheral blood, activated them by adding different maturation-inducing compounds, and cocultured them with T cells and HIV-1. We found that MDCs enhance HIV-1 infection through capture of the virus and subsequent transmission to T cells, and that differently matured MDC subsets have different HIV-1 transmission efficiencies. These differences were not due to soluble factors, viral capture differences, or the expression of integrins ICAM-1, -2, -3, or LFA-1. In contrast, regardless of their state of maturation, PDCs inhibit HIV-1 replication in T cells through the secretion of IFNalpha and an additional, unidentified small molecule. This study shows that the 2 main types of DCs have opposing roles in HIV-1 infection of T cells.

• Histatin 5-derived peptide with improved fungicidal properties enhances human immunodeficiency virus type 1 replication by promoting viral entry.

  Authors: Fedde Groot, Rogier W Sanders, Olivier Ter Brake, Kamran Nazmi, Enno C I Veerman, Jan G M Bolscher, Ben Berkhout

  Journal of virology. 10/2006; 80(18):9236-43.

  Antimicrobial peptides are found in a number of body compartments and are secreted at mucosal surfaces, where they form part of the innate immune system. Many of these small peptides have a broadAntimicrobial peptides are found in a number of body compartments and are secreted at mucosal surfaces, where they form part of the innate immune system. Many of these small peptides have a broad spectrum of inhibitory activity against bacteria, fungi, parasites, and viruses. Generally, the peptide's mode of action is binding and disruption of membranes due to its amphipathic properties. Histatin 5 is a salivary peptide that inhibits Candida albicans, an opportunistic fungus that causes oropharyngeal candidiasis in a majority of human immunodeficiency virus type 1 (HIV-1)-infected patients progressing towards AIDS. Previously, we increased the fungicidal properties of histatin 5 by replacing amino acids in the active domain of histatin 5 (Dh-5) (A. L. Ruissen, J. Groenink, E. J. Helmerhorst, E. Walgreen-Weterings, W. van't Hof, E. C. Veerman, and A. V. Nieuw Amerongen, Biochem. J. 356:361-368, 2001). In the current study, we tested the anti-HIV-1 activity of Dh-5 and its derivatives. Although Dh-5 inhibited HIV-1 replication, none of the peptide variants were more effective in this respect. In contrast, one of the derivatives, Dhvar2, significantly increased HIV-1 replication by promoting the envelope-mediated cell entry process. Most likely, Dhvar2 affects membranes, thereby facilitating fusion of viral and cellular membranes. This study shows that modification of antimicrobial peptides in order to improve their activity against a pathogen may have unpredictable and unwanted side effects on other pathogens.

• Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1.

  Authors: Fedde Groot, Taco W Kuijpers, Ben Berkhout, Esther C de Jong

  Retrovirology. 02/2006; 3:75.

  BACKGROUND: Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4+ T cells. We have demonstratedBACKGROUND: Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4+ T cells. We have demonstrated before that DC subsets expressing higher levels of intercellular adhesion molecule-1 (ICAM-1) are better HIV-1 transmitters. ICAM-1 binds leukocyte function-associated molecule-1 (LFA-1) on T cells, an integrin responsible for adhesion and signaling at the immunological synapse. To corroborate the importance of the ICAM-1- LFA-1 interaction, we performed transmission experiments to LFA-1 negative leukocytes from Leukocyte Adhesion Deficiency type 1 (LAD-1) patients. RESULTS: We clearly show that DC-mediated HIV-1 transmission to LAD-1 T cells is impaired in comparison to healthy controls. Furthermore, HIV-1 transmission to T cells from a unique LAD-1 patient with a well characterized LFA-1 activation defect was impaired as well, demonstrating that activation of LFA-1 is crucial for efficient transmission. Decreased cell adhesion between DC and LAD-1 T cells could also be illustrated by significantly smaller DC-T cell clusters after HIV-1 transmission. CONCLUSION: By making use of LFA-1 defect cells from unique patients, this study provides more insight into the mechanism of HIV-1 transmission by DC. This may offer new treatment options to reduce sexual transmission of HIV-1.

• Differential susceptibility of naïve, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission.

  Authors: Fedde Groot, Toni M M van Capel, Joost Schuitemaker, Ben Berkhout, Esther C de Jong

  Retrovirology. 02/2006; 3:52.

  BACKGROUND: Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1 by capture of the virus in the mucosa and subsequent transmission to CD4+ T cells. Several T cellBACKGROUND: Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1 by capture of the virus in the mucosa and subsequent transmission to CD4+ T cells. Several T cell subsets can be identified in humans: naïve T cells (TN) that initiate an immune response to new antigens, and memory T cells that respond to previously encountered pathogens. The memory T cell pool comprises central memory (TCM) and effector memory cells (TEM), which are characterized by distinct homing and effector functions. The TEM cell subset, which can be further divided into effector Th1 and Th2 cells, has been shown to be the prime target for viral replication after HIV-1 infection, and is abundantly present in mucosal tissues. RESULTS: We determined the susceptibility of TN, TCM and TEM cells to DC-mediated HIV-1 transmission and found that co-receptor expression on the respective T cell subsets is a decisive factor for transmission. Accordingly, CCR5-using (R5) HIV-1 was most efficiently transmitted to TEM cells, and CXCR4-using (X4) HIV-1 was preferentially transmitted to TN cells. CONCLUSION: The highly efficient R5 transfer to TEM cells suggests that mucosal T cells are an important target for DC-mediated transmission. This may contribute to the initial burst of virus replication that is observed in these cells. TN cells, which are the prime target for DC-mediated X4 virus transmission in our study, are considered to inefficiently support HIV-1 replication. Our results thus indicate that DC may play a decisive role in the susceptibility of TN cells to X4 tropic HIV-1.

• Differential susceptibility of naïve, central memory and effector memory T cells to dendritic cell-mediated HIV-1 transmission

  Authors: Fedde Groot, van Capel Toni, Joost Schuitemaker, Ben Berkhout, de Jong Esther

  Retrovirology. 01/2006;

  Abstract Background Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1 by capture of the virus in the mucosa and subsequent transmission to CD4⁺T cells.Abstract Background Dendritic cells (DC) have been proposed to facilitate sexual transmission of HIV-1 by capture of the virus in the mucosa and subsequent transmission to CD4⁺T cells. Several T cell subsets can be identified in humans: naïve T cells (T_N) that initiate an immune response to new antigens, and memory T cells that respond to previously encountered pathogens. The memory T cell pool comprises central memory (T_CM) and effector memory cells (T_EM), which are characterized by distinct homing and effector functions. The T_EMcell subset, which can be further divided into effector Th1 and Th2 cells, has been shown to be the prime target for viral replication after HIV-1 infection, and is abundantly present in mucosal tissues. Results We determined the susceptibility of T_N, T_CMand T_EMcells to DC-mediated HIV-1 transmission and found that co-receptor expression on the respective T cell subsets is a decisive factor for transmission. Accordingly, CCR5-using (R5) HIV-1 was most efficiently transmitted to T_EMcells, and CXCR4-using (X4) HIV-1 was preferentially transmitted to T_Ncells. Conclusion The highly efficient R5 transfer to T_EMcells suggests that mucosal T cells are an important target for DC-mediated transmission. This may contribute to the initial burst of virus replication that is observed in these cells. T_Ncells, which are the prime target for DC-mediated X4 virus transmission in our study, are considered to inefficiently support HIV-1 replication. Our results thus indicate that DC may play a decisive role in the susceptibility of T_Ncells to X4 tropic HIV-1.

• Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1

  Authors: Fedde Groot, Taco Kuijpers, Ben Berkhout, de Jong Esther

  Retrovirology. 01/2006;

  Abstract Background Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4⁺T cells. WeAbstract Background Dendritic cells (DC) have been proposed to mediate sexual HIV-1 transmission by capturing the virus in the mucosa and subsequently presenting it to CD4⁺T cells. We have demonstrated before that DC subsets expressing higher levels of intercellular adhesion molecule-1 (ICAM-1) are better HIV-1 transmitters. ICAM-1 binds leukocyte function-associated molecule-1 (LFA-1) on T cells, an integrin responsible for adhesion and signaling at the immunological synapse. To corroborate the importance of the ICAM-1— LFA-1 interaction, we performed transmission experiments to LFA-1 negative leukocytes from Leukocyte Adhesion Deficiency type 1 (LAD-1) patients. Results We clearly show that DC-mediated HIV-1 transmission to LAD-1 T cells is impaired in comparison to healthy controls. Furthermore, HIV-1 transmission to T cells from a unique LAD-1 patient with a well characterized LFA-1 activation defect was impaired as well, demonstrating that activation of LFA-1 is crucial for efficient transmission. Decreased cell adhesion between DC and LAD-1 T cells could also be illustrated by significantly smaller DC-T cell clusters after HIV-1 transmission. Conclusion By making use of LFA-1 defect cells from unique patients, this study provides more insight into the mechanism of HIV-1 transmission by DC. This may offer new treatment options to reduce sexual transmission of HIV-1.

• Lewis X component in human milk binds DC-SIGN and inhibits HIV-1 transfer to CD4+ T lymphocytes.

  Authors: Marloes A Naarding, Irene S Ludwig, Fedde Groot, Ben Berkhout, Teunis B H Geijtenbeek, Georgios Pollakis, William A Paxton

  The Journal of clinical investigation. 12/2005; 115(11):3256-64.

  DC-specific ICAM3-grabbing non-integrin (DC-SIGN), which is expressed on DCs, can interact with a variety of pathogens such as HIV-1, hepatitis C, Ebola, cytomegalovirus, Dengue virus, Mycobacterium,DC-specific ICAM3-grabbing non-integrin (DC-SIGN), which is expressed on DCs, can interact with a variety of pathogens such as HIV-1, hepatitis C, Ebola, cytomegalovirus, Dengue virus, Mycobacterium, Leishmania, and Candida albicans. We demonstrate that human milk can inhibit the DC-SIGN-mediated transfer of HIV-1 to CD4+ T lymphocytes as well as viral transfer by both immature and mature DCs. The inhibitory factor directly interacted with DC-SIGN and prevented the HIV-1 gp120 envelope protein from binding to the receptor. The human milk proteins lactoferrin, alpha-lactalbumin, lysozyme, beta-casein, and secretory leukocyte protease inhibitor did not bind DC-SIGN or demonstrate inhibition of viral transfer. The inhibitory effect could be fully alleviated with an Ab recognizing the Lewis X (LeX) sugar epitope, commonly found in human milk. LeX in polymeric form or conjugated to protein could mimic the inhibitory activity, whereas free LeX sugar epitopes could not. We reveal that a LeX motif present in human milk can bind to DC-SIGN and thereby prevent the capture and subsequent transfer of HIV-1 to CD4+ T lymphocytes. The presence of such a DC-SIGN-binding molecule in human milk may both influence antigenic presentation and interfere with pathogen transfer in breastfed infants.

• Lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type 1 transmission by blocking the DC-SIGN--gp120 interaction.

  Authors: Fedde Groot, Teunis B H Geijtenbeek, Rogier W Sanders, Christopher E Baldwin, Marta Sanchez-Hernandez, René Floris, Yvette van Kooyk, Esther C de Jong, Ben Berkhout

  Journal of virology. 04/2005; 79(5):3009-15.

  One of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, ofOne of the cell types first encountered by human immunodeficiency virus type 1 (HIV-1) following sexual transmission are dendritic cells (DC). DC capture HIV-1 through C-type lectin receptors, of which the best studied example is DC-SIGN, which mediates HIV-1 internalization. DC can keep the virus infectious for several days and are able to transmit HIV-1 to CD4(+) T cells. We tested proteins from milk and serum for their ability to block DC-mediated HIV-1 transmission, of which bovine lactoferrin (bLF) is the most potent inhibitor. bLF binds strongly to DC-SIGN, thus preventing virus capture and subsequent transmission. Interestingly, bLF is a much more efficient inhibitor of transmission than human lactoferrin. Since bLF is nontoxic and easy to purify in large quantities, it is an interesting candidate microbicide against HIV-1. Another advantage of bLF is its ability to block HIV-1 replication in T cells. DC-mediated capture of a bLF-resistant HIV-1 variant that was selected during long-term culturing in T cells could still be blocked by bLF. This underscores the usefulness of bLF as a microbicide drug to prevent HIV-1 transmission.

• The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

  Authors: R.W. Sanders, E. van Anken, A.A. Nabatov, I. Marije Liscaljet, I. Bontjer, D. Eggink, M Melchers, E. Busser, Martijn M. Dankers, Fedde Groot, L.J. Braakman, Ben Berkhout, W A Paxton

  Background: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120Background: The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain. Results: The 386 carbohydrate was not essential for folding of wt gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies. Conclusion: The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.