Ines Frank

HIV-1 subverts antigen processing in dendritic cells (DCs) resulting in viral uptake, infection, and transfer to T cells. Although DCs bound monomeric gp120 and HIV-1 similarly, virus rarely colocalized with endolysosomal markers, unlike gp120, suggesting HIV-1 alters endolysosomal trafficking. Virus within DC intracellular compartments rapidly moved to DC-CD4+ lymphocyte synapses when introduced to CD4+ lymphocyte cultures. Although viral harboring and transfer from nonlysosomal compartments was transient, given DC-associated virus protein, nucleic acids, and infectious HIV-1 transfer to CD4+, lymphocytes decayed within 24 hours. However a second long-term transfer phase was apparent in immature DCs after 48 hours as a zidovudine-sensitive rise in proviral DNA. Therefore, DCs transfer HIV-1 to CD4+ lymphocytes in 2 distinct phases. Immature and mature DCs first divert virus from the endolysosomal pathway to the DC-T-cell synapse. Secondly, the later transfer phase from immature DCs is through de novo HIV-1 production. Thus, the controversy of DCs being infected or not infected for the mechanics of viral transfer to CD4+ lymphocytes can be addressed as a function of time.

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell-specific intercellular adhesion molecule-grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3(+)HLA-DR(-) and CD3(-)HLA-DR(+) cells, with a significant proportion of the latter also expressing dendritic cell-specific intercellular adhesion molecule-grabbing integrin. Bead depletion studies demonstrated that such HLA-DR(+) cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.

OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.

As potential targets for human immunodeficiency virus type 1 and simian immunodeficiency virus (HIV-1 and SIV), dendritic cells (DCs) likely play a significant role in the onset and spread of infection as well as in the induction of antiviral immunity. Using the SIV-macaque system to study the very early events in DC-virus interactions, we compared chemically inactivated SIV having conformationally and functionally intact envelope glycoproteins (2,2'-dithiodipyridine [AT-2] SIV) to infectious and heat-treated SIV. Both human and macaque DCs interact similarly with SIV without detectable effects on DC viability, phenotype, or endocytic function. As assessed by measuring cell-associated viral RNA, considerable amounts of virus are captured by the DCs and this is reduced when the virus is heat treated or derived from a strain that expresses low levels of envelope glycoprotein. Immunostaining for SIV proteins and electron microscopy indicated that few intact virus particles are retained at the periphery of the endocytically active, immature DCs. This contrasts with a perinuclear localization of numerous virions in large vesicular compartments deeper within mature DCs (in which macropinocytosis is down-regulated). Both immature and mature DCs are capable of clathrin-coated pit-mediated uptake of SIV, supporting the notion that the receptor-mediated uptake of virus can occur readily in mature DCs. While large numbers of whole viruses were preferentially found in mature DCs, both immature and mature DCs contained similar amounts of viral RNA, suggesting that different uptake/virus entry mechanisms are active in immature and mature DCs. These findings have significant implications for cell-to-cell transmission of HIV-1 and SIV and support the use of AT-2 SIV, an authentic but noninfectious form of virus, as a useful tool for studies of processing and presentation of AT-2 SIV antigens by DCs.

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ (high)) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ (high) CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ (high)CD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.