HIV induces maturation of monocyte-derived dendritic cells and Langerhans cells

In HIV infection, dendritic cells (DCs) may play multiple roles, probably including initial HIV uptake in the anogenital mucosa, transport to lymph nodes, and subsequent transfer to T cells. The effects of HIV-1 on DC maturation are controversial, with several recent conflicting reports in the literature. In this study, microarray studies, confirmed by real-time PCR, demonstrated that the genes encoding DC surface maturation markers were among the most differentially expressed in monocyte-derived dendritic cells (MDDCs), derived from human blood, treated with live or aldrithriol-2-inactivated HIV-1(BaL). These effects translated to enhanced cell surface expression of these proteins but differential expression of maturation markers was only partial compared with the effects of a conventional potent maturation stimulus. Such partially mature MDDCs can be converted to fully mature cells by this same potent stimulus. Furthermore, live HIV-1 stimulated greater changes in maturation marker surface expression than aldrithriol-2-inactivated HIV-1 and this enhanced stimulation by live HIV-1 was mediated via CCR5, thus suggesting both viral replication-dependent and -independent mechanisms. These partially mature MDDCs demonstrated enhanced CCR7-mediated migration and are also able to stimulate interacting T cells in a MLR, suggesting DCs harboring HIV-1 might prepare CD4 lymphocytes for transfer of HIV-1. Increased maturation marker surface expression was also demonstrated in native DCs, ex vivo Langerhans cells derived from human skin. Thus, HIV initiates maturation of DCs which could facilitate subsequent enhanced transfer to T cells.     

Human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes

Recent epidemiologic studies show increasing human immunodeficiency virus type 1 (HIV-1) transmission through oral-genital contact. This paper examines the possibility that normal human oral keratinocytes (NHOKs) might be directly infected by HIV or might convey infectious HIV virions to adjacent leukocytes. PCR analysis of proviral DNA constructs showed that NHOKs can be infected by CXCR4-tropic (NL4-3 and ELI) and dualtropic (89.6) strains of HIV-1 to generate a weak but productive infection. CCR5-tropic strain Ba-L sustained minimal viral replication. Antibody inhibition studies showed that infection by CXCR4-tropic viral strains is mediated by the galactosylceramide receptor and the CXCR4 chemokine coreceptor. Coculture studies showed that infectious HIV-1 virions can also be conveyed from NHOKs to activated peripheral blood lymphocytes, suggesting a potential role of oral epithelial cells in the transmission of HIV infection.     

Mechanisms of nonrandom human immunodeficiency virus type 1 infection and double infection: preference in virus entry is important but is not the sole factor

We previously demonstrated that human immunodeficiency virus type 1 (HIV-1) infection is nonrandom and that double infection occurs more frequently than predicted from random events. To probe the possible mechanisms for nonrandom infection, we examined the role of HIV-1 entry pathways by using viruses pseudotyped with either CCR5-tropic HIV-1 Env or vesicular stomatitis virus G protein (VSV G). These two proteins use different receptors and entry pathways. We found that regardless of the protein used, double infection occurred more frequently than random events, indicating nonrandom HIV-1 infection in both entry pathways. However, the frequency of double infection differed significantly, depending on the envelope protein. In primary CD4(+) T cells, double infection occurred most frequently when both viruses had CCR5-tropic HIV-1 Env and least frequently when the two viruses had different envelopes. These results indicated that the preference in virus entry was a significant but not the only factor contributing to nonrandom double infection. Furthermore, we demonstrated that the CD4 expression level in primary T cells affects their susceptibility to CCR5-tropic HIV-1 infection but not VSV G-pseudotyped HIV-1 infection. We have also examined infection with two viruses pseudotyped with CCR5- or CXCR4-tropic HIV-1 Env and have found that double infection occurred more frequently than random events. These results indicate that coreceptor usage is not a barrier to recombination between the two virus populations. In our previous study, we also demonstrated nonrandom double infection via dendritic cell (DC)-mediated HIV-1 transmission. To test our hypothesis that multiple HIV-1 virions are transmitted during DC-T-cell contact, we used two populations of DCs, each capturing one vector virus, and added both DC populations to T cells. We observed a decreased frequency of double infection compared with experiments in which DCs captured both viruses simultaneously. Therefore, these results support our hypothesis that multiple virions are transmitted from DCs to T cells during cell-mediated HIV-1 transmission.     

Infection of specific dendritic cells by CCR5-tropic human immunodeficiency virus type 1 promotes cell-mediated transmission of virus resistant to broadly neutralizing antibodies

The tropism of human immunodeficiency virus type 1 for chemokine receptors plays an important role in the transmission of AIDS. Although CXCR4-tropic virus is more cytopathic for T cells, CCR5-tropic strains are transmitted more frequently in humans for reasons that are not understood. Phenotypically immature myeloid dendritic cells (mDCs) are preferentially infected by CCR5-tropic virus, in contrast to mature mDCs, which are not susceptible to infection but instead internalize virus into a protected intracellular compartment and enhance the infection of T cells. Here, we define a mechanism to explain preferential transmission of CCR5-tropic viruses based on their interaction with mDCs and sensitivity to neutralizing antibodies. Infected immature mDCs differentiated normally and were found to enhance CCR5-tropic but not CXCR4-tropic virus infection of T cells even in the continuous presence of neutralizing antibodies. Infectious synapses also formed normally in the presence of such antibodies. Infection of immature mDCs by CCR5-tropic virus can therefore establish a pool of infected cells that can efficiently transfer virus at the same time that they protect virus from antibody neutralization. This property of DCs may enhance infection, contribute to immune evasion, and could provide a selective advantage for CCR5-tropic virus transmission.     

Dendritic cells preferentially transfer CXCR4-using human immunodeficiency virus type 1 variants to CD4+ T lymphocytes in trans

Human immunodeficiency virus type 1 (HIV-1) preferentially utilizes the CCR5 coreceptor for target cell entry in the acute phase of infection, while later in disease progression the virus switches to the CXCR4 coreceptor in approximately 50% of patients. In response to HIV-1 the adaptive immune response is triggered, and antibody (Ab) production is elicited to block HIV-1 entry. We recently determined that dendritic cells (DCs) can efficiently capture Ab-neutralized HIV-1, restore infectivity, and transmit infectious virus to target cells. Here, we tested the effect of Abs on trans transmission of CCR5 or CXCR4 HIV-1 variants. We observed that transmission of HIV-1 by immature as well as mature DCs was significantly higher for CXCR4- than CCR5-tropic viral strains. Additionally, neutralizing Abs directed against either the gp41 or gp120 region of the envelope such as 2F5, 4E10, and V3-directed Abs inhibited transmission of CCR5-tropic HIV-1, whereas Ab-treated CXCR4-tropic virus demonstrated unaltered or increased transmission. To further study the effects of coreceptor usage we tested molecularly cloned HIV-1 variants with modifications in the envelope that were based on longitudinal gp120 V1 and V3 variable loop sequences from a patient progressing to AIDS. We observed that DCs preferentially facilitated infection of CD4⁺ T lymphocytes of viral strains with an envelope phenotype found late in disease. Taken together, our results illustrate that DCs transmit CXCR4-tropic HIV-1 much more efficiently than CCR5 strains; we hypothesize that this discrimination could contribute to the in vivo coreceptor switch after seroconversion and could be responsible for the increase in viral load.     

Compensatory link between fusion and endocytosis of human immunodeficiency virus type 1 in human CD4 T lymphocytes

Virions of the type 1 human immunodeficiency virus (HIV-1) can enter target cells by fusion or endocytosis, with sharply different functional consequences. Fusion promotes productive infection of the target cell, while endocytosis generally leads to virion inactivation in acidified endosomes or degradation in lysosomes. Virion fusion and endocytosis occur equally in T cells, but these pathways have been regarded as independent because endocytosis of HIV virions requires neither CD4 nor CCR5/CXCR4 engagement in HeLa-CD4 cells. Using flow cytometric techniques to assess the binding and entry of green fluorescent protein (GFP)-Vpr-labeled HIV virions into primary peripheral blood mononuclear cells, we have found that HIV fusion and endocytosis are restricted to the CD4-expressing subset of cells and that both pathways commonly require the initial binding of HIV virions to surface CD4 receptors. Blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry via the endocytic pathway. Similarly, inhibition of endosome acidification with bafilomycin A1, concanamycin A, or NH(4)Cl enhanced entry via the fusion pathway. Although fusion remained dependent on CD4 and chemokine receptor binding, the endosome inhibitors did not alter surface expression of CD4 and CXCR4. These results suggest that fusion in the presence of the endosome inhibitors likely occurs within nonacidified endosomes. However, the ability of these inhibitors to impair vesicle trafficking from early to late endosomes in some cells could also increase the recycling of these virion-containing endosomes to the cell surface, where fusion occurs. In summary, our results reveal an unexpected, CD4-mediated reciprocal relationship between the pathways governing HIV virion fusion and endocytosis.     

Double-stranded RNA stimulation or CD40 ligation of monocyte-derived dendritic cells as models to study their activation and maturation process

Monocyte-derived dendritic cells (DCs) were used as an in vitro model of myeloid DCs in order to determine a minimum marker pattern with which to characterize and distinguish different stages of DC activation and maturation. Phenotypic changes induced on immature DCs by two prototypic stimuli, poly I:C and CD40 ligation, were first examined. Both elicited HLA-DR, CD40, CD86 and CXCR4 upregulation, and CCR5 downregulation, but only CD40 ligand-stimulated DCs became CD83(+)\CCR7(+), whereas poly I:C-stimulated DCs expressed lower CD83 levels and were mostly CCR7(--). CD40 ligation and poly I:C elicited increased production of inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, of IL-10 and the CCL5 chemokine, but profiles differed as to higher IL-10, IL-12 and CCL22 (a CCR4 ligand important for T cell recruitment) levels for the former, and of CCL4 and CCL5 for the latter. Thus, a limited set of phenotypic markers, cytokine and chemokine production assays, may be used to distinguish the three stages in the life of DCs: immaturity, activation and full maturation. The ability of purified protein derivative-loaded DCs to stimulate autologous T cells to produce IL-2, IL-4 and interferon-gamma indeed depended on their activation stage and endocytic activity, which decreased upon maturation. We then examined whether ligation of CD4, CCR5 and\or CXCR4, the receptor and coreceptors of human immunodeficiency virus envelope gp120, respectively, affected DC activation or maturation, neither a monoclonal antibody to the gp120-binding site on CD4 nor CCL5 nor CXCL12, the natural ligands of CCR5 and CXCR4, respectively, nor gp120 altered the DC activation and maturation processes.     

HIV-dendritic cell interactions promote efficient viral infection of T cells

Dendritic cells (DC) are bone marrow-derived leukocytes that act as powerful stimulators of primary and secondary immune responses. Langerhans cells (LC), which are immature DC in epidermis and genital mucosa, are generally believed to be the initial cells infected with HIV following mucosal exposure to virus. Interestingly, freshly isolated LC express the HIV coreceptor CCR5, but not CXCR4, on their cell surfaces. This expression pattern would theoretically allow only macrophage-tropic [and not T cell (TC)-tropic] HIV to be transmitted across intact mucosal epithelium. In vitro, it is known that HIV infects LC (and other DC) in a CD4- and HIV coreceptor-dependent manner. In addition, HIV can be captured by prominent stellate processes on the surface of LC/DC. HIV-infected DC, as well as DC that have captured HIV, efficiently transmit virus to TC during antigen-specific TC activation. Thus, DC may be involved in HIV plasma viremia increases observed following antigenic exposure, e.g. immunizations, in chronically HIV-infected individuals by (1) activating latently infected TC or (2) activating and transmitting virus to new target TC. In summary, DC most likely play a major role in primary HIV infection by allowing virus to breach mucosal surfaces, and can act during both initial and chronic phases of HIV disease by facilitating infection and depletion of TC.     

Natural alpha interferon-producing cells respond to human immunodeficiency virus type 1 with alpha interferon production and maturation into dendritic cells

Natural alpha interferon (IFN-alpha)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR(+) CD11c(-) lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-alpha production and their differentiation into DCs. After incubation with either NL4-3 or JR-CSF, IPCs produced a large amount of IFN-alpha and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-alpha production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-alpha production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.     

Maturation of blood-derived dendritic cells enhances human immunodeficiency virus type 1 capture and transmission

Dendritic cells (DCs) are specialized antigen-presenting cells. However, DCs exposed to human immunodeficiency virus type 1 (HIV-1) are also able to transmit a vigorous cytopathic infection to CD4(+) T cells, a process that has been frequently related to the ability of DC-SIGN to bind HIV-1 envelope glycoproteins. The maturation of DCs can increase the efficiency of HIV-1 transmission through trans infection. We aimed to comparatively study the effect of maturation in monocyte-derived DCs (MDDCs) and blood-derived myeloid DCs during the HIV-1 capture process. In vitro capture and transmission of envelope-pseudotyped HIV-1 and its homologous replication-competent virus to susceptible target cells were assessed by p24(gag) detection, luciferase activity, and both confocal and electron microscopy. Maturation of MDDCs or myeloid DCs enhanced the active capture of HIV-1 in a DC-SIGN- and viral envelope glycoprotein-independent manner, increasing the life span of trapped virus. Moreover, higher viral transmission of mature DCs to CD4(+) T cells was highly dependent on active viral capture, a process mediated through cholesterol-enriched domains. Mature DCs concentrated captured virus in a single large vesicle staining for CD81 and CD63 tetraspanins, while immature DCs lacked these structures, suggesting different intracellular trafficking processes. These observations help to explain the greater ability of mature DCs to transfer HIV-1 to T lymphocytes, a process that can potentially contribute to the viral dissemination at lymph nodes in vivo, where viral replication takes place and there is a continuous interaction between susceptible T cells and mature DCs.     

Differential Tropism and Replication Kinetics of Human Immunodeficiency Virus Type 1 Isolates in Thymocytes: Coreceptor Expression Allows Viral Entry, but Productive Infection of Distinct Subsets Is Determined at the Postentry Level

Human thymocytes are readily infected with human immunodeficiency virus type 1 (HIV-1) in vivo and in vitro. In this study, we found that the kinetics of replication and cytopathic effects of two molecular isolates, NL4-3 and JR-CSF, in postnatal thymocytes are best explained by the distribution of chemokine receptors used for viral entry. CXCR4 was expressed at high levels on most thymocytes, whereas CCR5 expression was restricted to only 0.1 to 2% of thymocytes. The difference in the amount of proviral DNA detected after infection of fresh thymocytes with NL4-3 or JR-CSF correlated with the levels of CXCR4 and CCR5 surface expression. Anti-CCR5 blocking studies showed that low levels of CCR5 were necessary and sufficient for JR-CSF entry in thymocytes. Interleukin-2 (IL-2), IL-4, and IL-7, cytokines normally present in the thymus, influenced the expression of CXCR4 and CCR5 on thymocytes and thus increased the infectivity and spread of both NL4-3 and JR-CSF in culture. NL4-3 was produced by both immature and mature thymocytes, whereas JR-CSF production was restricted to the mature CD1⁻/CD69⁺ population. Although CXCR4 and CCR5 distribution readily explained viral entry in mature CD69⁺ and immature CD69⁻ cells, and correlated with proviral DNA distribution, we found that viral production was favored in CD69⁺ cells. Therefore, while expression of CD4 and appropriate coreceptors are essential determinants of viral entry, factors related to activation and stage-specific maturation contribute to HIV-1 replication in thymocyte subsets. These results have direct implications for HIV-1 pathogenesis in pediatric patients.     

Advanced glycation end products inhibit both infection and transmission in trans of HIV-1 from monocyte-derived dendritic cells to autologous T cells

Highly active antiretroviral therapy is associated with carbohydrate metabolic alterations that may lead to diabetes. One consequence of hyperglycemia is the formation of advanced glycation end products (AGEs) that are involved in diabetes complications. We investigated the impact of AGEs on the infection of monocyte-derived dendritic cells (MDDCs) by HIV-1 and the ability of MDDCs to transmit the virus to T cells. We showed that AGEs could inhibit infection of MDDCs with primary R5-tropic HIV-1(Ba-L) by up to 85 ± 9.2% and with primary X4-tropic HIV-1(VN44) by up to 60 ± 8.5%. This inhibitory effect of AGEs was not prevented by a neutralizing anti-receptor for advanced glycation end products (anti-RAGE) Ab, demonstrating a RAGE-independent mechanism. Moreover, AGEs inhibited by 70-80% the transmission in trans of the virus to CD4 T cells. Despite the inhibitory effect of AGEs on both MDDC infection and virus transmission in trans, no inhibition of virus attachment to cell membrane was observed, confirming that attachment and transmission of the virus involve independent mechanisms. The inhibitory effect of AGEs on infection was associated with a RAGE-independent downregulation of CD4 at the cell membrane and by a RAGE-dependent repression of the CXCR4 and CCR5 HIV-1 receptors. AGEs induce the secretion of proinflammatory cytokines IL-6, TNF-α, and IL-12, but not RANTES or MIP-1α, and did not lead to MDDC maturation as demonstrated by the lack of expression of the CD83 molecule. Taken together, our results suggest that AGEs can play an inhibiting role in HIV-1 infection in patients who accumulate circulating AGEs, including patients treated with protease inhibitors that developed diabetes.     

Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide

BACKGROUND: The predominant mode of HIV-1 transmission is by heterosexual contact. The cervical/vaginal mucosa is the main port of HIV entry in women. A safe and effective topical microbicide against HIV is urgently needed to prevent sexual transmission. Hence, we evaluated griffithsin (GRFT), a 12.7 kDa carbohydrate-binding protein, both native and recombinant GRFT, potently inhibited both CXCR4-and CCR5-tropic HIV infection and transmission in vitro. METHODS: The antiviral efficacy of native and recombinant GRFT against CXCR4-and CCR5-tropic HIV and SHIV strains and SIVmac251 was evaluated by in vitro assays. We also evaluated the time course of antiviral activity and stability of GRFT in cervical/vaginal lavage as a function of pH 4-8. RESULTS: Griffithsin blocked CXCR4-and CCR5-tropic viruses at less than 1 nm concentrations and exhibited a high potency. GRFT was stable in cervical/vaginal lavage fluid and maintained a similar potency of anti-HIV activity. GRFT is not only a highly potent HIV entry inhibitor, but also prevents cell fusion and cell-to-cell transmission of HIV. CONCLUSIONS: The in vitro efficacy of GRFT revealed low cytotoxicity, high potency, rapid onset of antiviral activity and long-term stability in cervical/vaginal lavage. GRFT is an excellent candidate for anti-HIV microbicide development.     

Resolution of de novo HIV production and trafficking in immature dendritic cells

The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus of HIV p17 matrix within the HIV gag protein. Using this construct in combination with biarsenical dyes, we observed restricted staining of the dTCM to de novo-synthesized uncleaved gag in the DC cytosol. Co-staining with HIV gag antibodies, reactive to either p17 matrix or p24 capsid, preferentially stained mature virions and thus allowed us to track the virus at distinct stages of its life cycle within DCs and upon transfer to neighboring DCs or T cells. Thus, in staining HIV gag with biarsenical dye system in situ, we characterized a replication-competent virus capable of being tracked preferentially within infected leukocytes and observed in detail the dynamic nature of the HIV production and transfer in primary DCs.     

Opsonization of HIV with complement enhances infection of dendritic cells and viral transfer to CD4 T cells in a CR3 and DC-SIGN-dependent manner

In the present study, we demonstrated that opsonization of primary HIV-1 with human complement enhances infection of immature monocyte-derived dendritic cells (iDC) and transmission in trans of HIV to autologous CD4(+) T lymphocytes. Infection of iDC by opsonized primary R5- and X4-tropic HIV was increased 3- to 5-fold as compared with infection by the corresponding unopsonized HIV. Enhancement of infection was dependent on CR3 as demonstrated by inhibition induced by blocking Abs. The interaction of HIV with CCR5 and CXCR4 on iDC was affected by opsonization. Indeed, stromal-derived factor-1 was more efficient in inhibiting infection of iDC with opsonized R5-tropic HIV-1(BaL) (45%) than with heat-inactivated complement opsonized virus and similarly RANTES inhibited more efficiently infection of iDC with opsonized X4-tropic HIV-1(NDK) (42%) than with heat-inactivated complement opsonized virus. We also showed that attachment of complement-opsonized virus to DC-specific ICAM-grabbing nonintegrin (DC-SIGN) molecule on iDC and HeLa DC-SIGN(+) CR3(-) cells was 46% and 50% higher compared with heat-inactivated complement opsonized virus, respectively. Hence, Abs to DC-SIGN suppressed up to 80% and 60% the binding of opsonized virus to HeLa cells and iDC, respectively. Furthermore, Abs to DC-SIGN inhibited up to 70% of the infection of iDC and up to 65% of infection in trans of autologous lymphocytes with opsonized virus. These results further demonstrated the role of DC-SIGN in complement opsonized virus uptake and infection. Thus, the virus uses complement to its advantage to facilitate early steps leading to infection following mucosal transmission of HIV.     

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.     

Rabbit Cells Expressing Human CD4 and Human CCR5 Are Highly Permissive for Human Immunodeficiency Virus Type 1 Infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

HIV-1-induced migration of monocyte-derived dendritic cells is associated with differential activation of MAPK pathways

From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3beta (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4(+) cells.     

Infection of dendritic cells (DCs), not DC-SIGN-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to T cells

The C-type lectin DC-SIGN expressed on immature dendritic cells (DCs) captures human immunodeficiency virus (HIV) particles and enhances the infection of CD4+ T cells. This process, known as trans-enhancement of T-cell infection, has been related to HIV endocytosis. It has been proposed that DC-SIGN targets HIV to a nondegradative compartment within DCs and DC-SIGN-expressing cells, allowing incoming virus to persist for several days before infecting target cells. In this study, we provide several lines of evidence suggesting that intracellular storage of intact virions does not contribute to HIV transmission. We show that endocytosis-defective DC-SIGN molecules enhance T-cell infection as efficiently as their wild-type counterparts, indicating that DC-SIGN-mediated HIV internalization is dispensable for trans-enhancement. Furthermore, using immature DCs that are genetically resistant to infection, we demonstrate that several days after viral uptake, HIV transfer from DCs to T cells requires viral fusion and occurs exclusively through DC infection and transmission of newly synthesized viral particles. Importantly, our results suggest that DC-SIGN participates in this process by cooperating with the HIV entry receptors to facilitate cis-infection of immature DCs and subsequent viral transfer to T cells. We suggest that such a mechanism, rather than intracellular storage of incoming virus, accounts for the long-term transfer of HIV to CD4+ T cells and may contribute to the spread of infection by DCs.