Human immunodeficiency virus type 1 (HIV-I) envelope (Env), responsible for both receptor binding and membrane fusion, consists of a gpl20 surface subunit noncovalently associated with a gp41 membrane bound subunit. It is the gpl20 subunit that binds and interacts with CD4 and a coreceptor prior to entrance of the virus into a cell. In addition to the primary receptor and coreceptor required for viral entry, viral attachment molecules have been described which could modulate the efficiency of the viral entry process. The recent discovery of a calcium-dependent (C-type) lectin, DC-SIGN, which binds to monomeric HIV Env gpl20 with a greater affinity than CD4, prompted us to re-examine the nature of this viral attachment process and its contribution to the process of viral entry. It has been hypothesized that a crucial step in the establishment of primary HIV infection is the transfer of virus from dendritic cells (DCs) in the submucosa to permissive T-cells in secondary lymphoid organs. The relatively specific expression of DC-SIGN on DCs, and its proposed role as a conduit for this transfer of HIV underscores the need to understand further the biology of this process. Indeed, DC-SIGN may have additional functions that have been heretofore unrecognized. For example, we have found that DC-SIGN expression in cis, that is, on permissive cells that express CD4 and coreceptor, markedly enhanced the efficiency of viral entry. Thus, DC-SIGN expression in-cis allows viral entry via vanishing levels of co-receptor or via use of alternate co-receptors that are otherwise used very inefficiently. In addition, it has also been reported that mRNA coding for putative soluble isoforms of DC-SIGN can be found in primary tissues. It is the driving hypothesis of this proposal that DC-SlGN can efficiently facilitate H1V/SIV infection in-trans and in-cis. The overall goal of this proposal is to gain a better understanding of DC-SIGN's biology and function and lay the foundation to better evaluate its putative role in viral transmission and pathogenesis in vivo. Understanding the structural basis for DC-SIGN/ gpl20 interaction will also inform efforts to design novel immunogens that might elicit antibodies that neutralize DC-SIGN/gpl20 binding. In this proposal, we will pursue three Specific Aims to (1) Examine the nature of enhanced viral entry and viral transfer when DC-SIGN is expressed in cis and in trans, respectively, (2) Determine the structural and mechanistic underpinnings of DC-SlGN/ HIV gpl20 interactions, and (3) Study the biology of DC-SIGN in vivo using two models for studying HIV pathogenesis in the gut mucosa and thymus, respectively.
