Mechanism(s) promoting HIV-1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-cultures

Resting CD4(+) T cells in the lymphoid tissue (LT) are essential producers of virions at the beginning of HIV infection in vivo. We previously developed a model that allowed in vitro infection of non-prestimulated T lymphocytes in the presence of autologous B lymphocytes and complement. In this study, we try to clarify the mechanism(s) responsible for virus transmission in unstimulated autologous B cell/T cell co-cultures. Ex vivo analyses of patient plasma samples revealed that HIV was opsonized. Flow cytometry showed that opsonized virus preferentially bound to complement receptor (CR)-2 on B lymphocytes in primary B cell/T cell co-cultures. As indicated by cytokine measurements and transwell experiments, soluble factors seemed to play a minor role in enabling infection. Rather, direct interaction between B and T lymphocytes and direct binding of opsonized virus to CR2 on B cells turned out to be essential for productive infection. Antibodies blocking cell-cell adhesion inhibited p24 antigen production. An anti-CR2 antibody blocking C3d-CR2 binding also significantly reduced viral replication. Since the infection of unstimulated T cells by opsonized primary HIV isolates in the presence of B cells was highly efficient independent of the tropism of the virus, this mechanism may be critical in the pathogenesis of HIV.     

Mannose binding lectin (MBL) and HIV

The envelope protein (gp120/gp41) of HIV-1 is highly glycosylated with about half of the molecular mass of gp120 consisting of N-linked carbohydrates. While glycosylation of HIV gp120/gp41 provides a formidable barrier for development of strong antibody responses to the virus, it also provides a potential site of attack by the innate immune system through the C-type lectin mannose binding lectin (MBL) (also called mannan binding lectin or mannan binding protein). A number of studies have clearly shown that MBL binds to HIV. Binding of MBL to HIV is dependent on the high-mannose glycans on gp120 while host cell glycans incorporated into virions do not contribute substantially to this interaction. It is notable that MBL, due to its specificity for the types of glycans that are abundant on gp120, has been shown to interact with all tested HIV strains. While direct neutralization of HIV produced in T cell lines by MBL has been reported, neutralization is relatively low for HIV primary isolates. However, drugs that alter processing of carbohydrates enhance neutralization of HIV primary isolates by MBL. Complement activation on gp120 and opsonization of HIV due to MBL binding have also been observed but these immune mechanisms have not been studied in detail. MBL has also been shown to block the interaction between HIV and DC-SIGN. Clinical studies show that levels of MBL, an acute-phase protein, increase during HIV disease. The effects of MBL on HIV disease progression and transmission are equivocal with some studies showing positive effects and other showing no effect or negative effects. Because of apparently universal reactivity with HIV strains, MBL clearly represents an important mechanism for recognition of HIV by the immune system. However, further studies are needed to define the in vivo contribution of MBL to clearance and destruction of HIV, the reasons for low neutralization by MBL and ways that MBL anti-viral effects can be augmented.     

Evidence for the Role of CR1 (CD35), in addition to CR2 (CD21), in Facilitating Infection of Human T Cells with Opsonized HIV

Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor-bearing target cells. We have studied complement-mediated enhancement of infection of the human CD4-positive T-cell line HPB-ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4-positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3- to 10-fold enhancement of infection of HPB-ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross-linked anti-CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement-mediated infection by 50–80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement-mediated enhancement of infection was observed with HIV-1 Bru but not with HIV-1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor-expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain.     

HIV-1 induced generation of C5a attracts immature dendriticcells and promotes infection of autologous T cells

For the recruitment of dendritic cells (DC) to the site of infection, DC express several sensors for danger signals, such as receptors for C5a. This anaphylatoxin is generated upon complement activation. As HIV-1 triggers the complement cascade, we determined whether C5a is generated by the virus and tested the functional activity of C5a in migration and infection assays. The immature (i)DC responded in migration assays to recombinant C5a and native C5a, which was generated in situ upon activation of the complement system by HIV-1. In combined migration and infection assays, a C5a-dependent enhancement of HIV-1 infection in DC-T cell cocultures was observed. These results indicate that HIV induces generation of C5a and thereby attracts iDC, which in turn promote the productive infection of autologous primary T cells.     

Blocking of integrins inhibits HIV‐1 infection of human cervical mucosa immune cells with free and complement‐opsonized virions

The initial interaction between HIV‐1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV‐1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV‐1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV‐1 infection of dendritic cells (DCs) compared with that by free HIV‐1, but this increased infection was not observed with CD4⁺ T cells. Blockage of the α4‐, β7‐, and β1‐integrins significantly inhibited HIV‐1 infection of both DCs and CD4⁺ T cells. We found a greater impairment of HIV‐1 infection in DCs for complement‐opsonized virions compared with that of free virions when αM/β2‐ and α4‐integrins were blocked. Blocking the C‐type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4⁺ T‐cell infection. We show that blocking of integrins decreases the HIV‐1 infection of both mucosal DCs and CD4⁺ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV‐1 infection.     

Complement-opsonized HIV: the free rider on its way to infection

The complement system (C) is one of the main humoral components of innate immunity. Three major tasks of C against invading pathogens are: (i) lysis of pathogens by the formation of the membrane attack complex (MAC); (ii) opsonization of pathogens with complement fragments to favor phagocytosis; and (iii) attraction of inflammatory cells by chemotaxis. Like other particles, HIV activates C and becomes opsonized. To escape complement-mediated lysis, HIV has adopted various properties, which include the acquisition of HIV-associated molecules (HAMs) belonging to the family of complement regulators, such as CD46, CD55, CD59, and the interaction with humoral regulatory factors like factor H (fH). Opsonized virus may bind to complement receptor positive cells to infect them more efficiently or to remain bound on the surface of such cells. In the latter case HIV can be transmitted to cells susceptible for infection. This review discusses several aspects of C-HIV interactions and provides a model for the dynamics of this process.     

Inhibition of DC-SIGN-mediated trans infection of T cells by mannose-binding lectin

Some dendritic cells (DC) express a cell-surface lectin called 'dendritic cell-specific intracellular adhesion molecule 3 (ICAM-3)-grabbing non-integrin' (DC-SIGN). DC-SIGN has been shown to mediate a type of infection called 'trans' infection, where DC bind human immunodeficiency virus (HIV) and efficiently transfer the virus to T cells. We investigated the possibility that mannose-binding lectin (MBL), a soluble lectin that functions as a recognition molecule in innate immunity and that binds to HIV, could block trans infection mediated by DC-SIGN. Binding studies with glycoprotein (gp)120/gp41-positive and -negative virus preparations suggested that DC-SIGN and MBL bind primarily to glycans on gp120/gp41, as opposed to glycans on host-cell-derived proteins, indicating a close overlap in the binding site of the two lectins and supporting the notion that MBL could prevent binding of HIV to DC-SIGN. Preincubation of X4, R5 or dual-tropic HIV strains with MBL prevented DC-SIGN-mediated trans infection of T cells. The mechanism of MBL blocking trans infection of T cells was at least partly caused by blocking of virus binding to DC-SIGN positive cells. This study shows that MBL prevents DC-SIGN-mediated trans infection of T cells in vitro and suggests that in infected persons, MBL may inhibit DC-SIGN-mediated uptake and spread of HIV.     

Enhanced human immunodeficiency virus infection in macrophages by high-molecular-weight dextran sulfate is associated with conformational changes of gp120 and expression of the CCR5 receptor.

High-molecular-weight dextran sulfate (HMDS) inhibits infection of CD4+ lymphocytes by T-cell (T)-tropic human immunodeficiency virus (HIV) isolates, but augments replication of macrophage (M)-tropic isolates in primary human macrophages and phorbol myristate acetate (PMA)-differentiated THP-1 monocytic cells. To address the mechanism responsible for HMDS-mediated increases in HIV replication in macrophages, we analyzed the interaction between HMDS and functional domains of gp120 on the surface of PMA-differentiated THP-1 cells infected with M-tropic HIV isolates. Immunofluorescence staining of the infected cells revealed that HMDS inhibited the binding of monoclonal antibodies (mAbs) directed to the V3 and C4 domains of gp120, but augmented the binding of three neutralizing antibodies directed to the V2 region of gp120. The extent of HMDS-mediated changes within the V2 loop of gp120 was associated with increased virus binding and replication in PMA-differentiated THP-1 cells and primary macrophages. The effect was dependent on expression of the CCR5 receptor and was inhibited by the beta-chemokine RANTES. Results of this study suggest that HMDS-mediated increases in HIV infection in macrophages are associated with conformational changes within the V2 region of gp120 and enhanced interaction between gp120 and the CCR5 coreceptor on the target cell.     

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.     

Complement opsonization of HIV‐1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells

Induction of optimal HIV‐1‐specific T‐cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV‐1 in human monocyte‐derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV‐1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7‐integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV‐1 exposure; complement‐opsonized HIV‐1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7‐integrin can promote or disfavor antigen presentation probably by routing HIV‐1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV‐1 affects the antigen‐processing machinery.     

DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection

Fusion of the human immunodeficiency virus (HIV) to the plasma membrane of target cells is mediated by interaction of its envelope glycoprotein, gp120, with CD4 and appropriate chemokine receptors. gp120 additionally binds to DC-SIGN, a C-type lectin expressed on immature dendritic cells. This interaction does not result in viral fusion, but instead contributes to enhanced infection in trans of target cells that express CD4 and chemokine receptors. Here we show that DC-SIGN mediates rapid internalization of intact HIV into a low pH nonlysosomal compartment. Internalized virus retains competence to infect target cells. Removal of the DC-SIGN cytoplasmic tail reduced viral uptake and abrogated the trans-enhancement of T cell infection. We propose that HIV binds to DC-SIGN to gain access to an intracellular compartment that contributes to augmentation or retention of viral infectivity.     

Bitter-sweet symphony: defining the role of dendritic cell gp120 receptors in HIV infection.

BACKGROUND: Dendritic cells (DC) are believed to be one of the first cell types infected during HIV transmission. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte derived DC (MDDC) rather than CD4. The role of other CLRs in HIV binding and HIV binding by CLRs on other types of DC in vivo is largely unknown. OBJECTIVES AND STUDY DESIGN: Review HIV binding to DC populations, both in vitro and in vivo, in light of the immense interest of a recently re-identified CLR called DC-SIGN. RESULTS AND CONCLUSIONS: From recent work, it is clear that immature MDDC have a complex pattern of HIV gp120 binding. In contrast to other cell types gp120 has the potential to bind to several receptors on DC including CD4 and several types of C type lectin receptor, not just exclusively DC-SIGN. Given the diverse types of DC in vivo future work will need to focus on defining the receptors for HIV binding to these different cell types. Mucosal transmission of HIV in vivo targets immature sessile DCs, including Langerhans cells which lack DC-SIGN. The role of CLRs and DC-SIGN in such transmission remains to be defined.     

HIV and human complement: inefficient virolysis and effective adherence

Both, HIV envelope proteins gp120 and gp41 can directly activate complement system, even in the absence of HIV-specific antibodies. During the budding process HIV acquires host membrane-associated molecules among these complement regulatory proteins (CRPs). The presence of CRPs on the viral surface rescues HIV from complement-mediated virolysis. The inefficient virolysis results in the deposition of complement-fragments on the viral surface allowing interactions of HIV with complement receptor expressing cells. In this review, the interaction of HIV with the complement system and the consequences of complement opsonisation on virus infection will be discussed.     

CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4.

Peripheral blood and tissue mononuclear phagocytes serve as major viral reservoirs in HIV-infected individuals. We investigated the role of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) in mediating productive infection with complement-opsonized HIV-1 and HIV-2 of cultured normal human peripheral blood monocytes, the promonocytic cell line THP-1, the monocytic cell line Mono Mac 6 and the glial cell line U251-MG. Cells were infected with the HTLV-IIIB strain of HIV-1 or the LAV-2 strain of HIV-2 that had been preopsonized with fresh human normal HIV seronegative serum. Productive infection was assessed by syncytia formation, the MTT cytotoxicity assay and/or release of p24 antigen in culture supernatants. Using suboptimal amounts of virus to infect the cells, we observed a higher and earlier productive infection of the cells with complement-opsonized HIV than with unopsonized virus. The enhancing effect of complement was totally suppressed by blocking CR1 or CR3 function with F(ab)'2 fragments of anti-receptor MoAbs; while blocking of the LFA-1 antigen had no effect. The infection of monocytic cells with complement-opsonized virus occurred independently of CD4 since it was not inhibited by F(ab)'2 fragments of a MoAb against the gp120 binding site of CD4 and since infection also occurred with Mono Mac 6 and U251-MG cells, which lack expression of the CD4 antigen and of CD4 mRNA. These observations suggest that complement may mediate productive infection of cells of the monocytic lineage with 'lymphocytotropic' HIV strains independently of CD4.     

Involvement of macrophage mannose receptor in the binding and transmission of HIV by macrophages

The human immunodeficiency virus (HIV) is an enveloped virus whose surface glycoprotein gp120 binds CD4 on target cell membranes to initiate infection. About half of the carbohydrates on gp120 are terminally mannosylated, a pattern common to many pathogens. We have examined the ability of macrophage mannose receptor (MMR) on primary monocyte-derived macrophages to bind HIV and facilitate its transmission to T cells. We adapted the tyramide signal amplification system for fluorescence detection of HIV bound to macrophages. Our data show that approximately 60% of the initial association of HIV with macrophages that lack expression of DC-SIGN (a dendritic cell-specific ICAM-3 receptor/HIV-1-binding protein) is MMR mediated, as evidenced by inhibition with mannan, D-mannose, EDTA, and soluble mannose-binding lectin, but not by D-galactose. This inhibition is not seen in cells that lack MMR. Macrophages are able to mediate transmission of bound HIV to co-cultured T cells, and this transmission is blocked up to 80% by inhibitors of MMR binding. Unlike virus bound to DC-SIGN, macrophage-bound HIV has a slightly lower half-life compared to free virus, with no transmission in co-culture observed beyond 24 h after virus binding to macrophages. Results obtained with endocytosis inhibitors indicate that this decrease in viral longevity is due to rapid internalization of macrophage-bound HIV. Together, these results suggest a substantial role for MMR in the binding and transmission of HIV-1 by macrophages.     

Conglutinin Binds The HIV-1 Envelope Glycoprotein gpl60 and Inhibits its Interaction with Cell Membrane CD4

The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160 was calcium-dependent, which is characteristic of the binding of a C-type lectin to its ligand, and the binding was inhibited in a dose-dependent manner with N-acetyl-D-glucosamine. Deglycosylation of rgp160 abrogated the conglutinin binding. In addition, conglutinin exerted a dose-dependent inhibition of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein.     

HIV-1 subverts the complement system in semen to enhance viral transmission

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.     

Receptor-mediated uptake of starch and mannan microparticles by macrophages: relative contribution of receptors for complement, immunoglobulins and carbohydrates

The contribution of different macrophage receptors to the uptake of polyacryl starch and polyacryl mannan microparticles by macrophages was studied. Both types of microparticle were taken up to a larger and similar extent when they had been pre-incubated with serum, indicating the importance of serum factors in the uptake process. These results were supported by organ distribution studies in mice, showing that the two microparticles were rapidly targeted to the liver and spleen after i.v. injection. The carbohydrate-specific mannose/fucose receptor was found to contribute significantly to the uptake of non-opsonized but not opsonized mannan microparticles. The two different microparticles were found to activate the alternative complement pathway and to adsorb immunoglobulin G, human serum albumin and fibronectin to their surface. Inhibition experiments provided evidence for the involvement of a complement receptor (CR3) and an Fc-receptor (FCR) for IgG in the uptake of opsonized microparticles.