This thesis describes an investigation of the mechanisms by which DC-SIGN mediates enhancement of HIV-1 infection. Mannose-binding C-type lectin receptors, expressed on Langerhans cells and subepithelial dendritic cells (DCs), play an important role in HIV-1capture and subsequent dissemination to lymph nodes. DC-SIGN mediates both productive infection of DCs and trans-infection of CD4+ T cells that occurs in the absence of replication. The molecular events involved in transmission have not been fully defined. In this study, surface plasmon resonance analyses demonstrated that DC-SIGN, but not langerin, increases the binding affinity of trimeric gp140 envelope glycoproteins to CD4. In vitro infection experiments demonstrated significantly lower enhancement of a CD4-independent compared with a CD4-dependent strain. DC-SIGN increased the relative rate of infection of the CD4-dependent strain but had no effect on the CD4- independent strain. These findings are consistent with a mechanism in which DC-SIGN binding to glycans of the HIV envelope protein increases exposure of the CD4 binding site to enhance infection. To investigate the specificity and nature of glycan binding required for enhancement of HIV infection, a chimaeric C-type lectin was prepared where the carbohydrate recognition domain (CRD) of DC-SIGN was substituted by the CRD of langerin. Enhancement of HIV infection was then determined in TZM-bl cells expressing DCSIGN, langerin or the chimaeric DC-SIGN/langerin lectin. All lectins significantly enhanced infection compared with TZM-bl cells (in the absence of lectin). These data suggest that the increased flexibility of the CRD associated with DC-SIGN does not contribute significantly to C-type lectin-mediated enhancement of infection. To determine whether DC-SIGN binding to specific glycans of the envelope protein is associated with enhanced infection, mutagenised HIV-1 virions with specific glycan deletions (at residues 386 and 397) were compared with the parental strain using an in vitro model of DC-SIGN mediated enhancement of infection. Deletion of the N-Linked glycan at residue 386 reduced the level of enhancement. These studies also provided some evidence that DC-SIGN binding to the HIV envelope protein may induce further perturbation in the structure leading to increased exposure of the co-receptor binding site.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677098 |
| Date | January 2015 |
| Creators | Scala, Carlo |
| Contributors | Kelly, Charles George ; Naglik, Julian Richard |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://kclpure.kcl.ac.uk/portal/en/theses/mechanism-of-dcsign-mediated-enhancement-of-hiv1-infection(1cf4fa70-8144-4828-810c-e28900c962bb).html |
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