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Dendritic cells (DCs) transmit human immunodeficiency virus type 1 (HIV-1) to CD4+ T cells through the trans- and cis-infection pathways; however, little is known about the relative efficiencies of these pathways and whether they are interdependent. Here we compare cis- and trans-infections of HIV-1 mediated by immature DCs (iDCs) and mature DCs (mDCs), using replication-competent and single-cycle HIV-1. Monocyte-derived iDCs were differentiated into various types of mDCs by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and CD40 ligand (CD40L). iDCs and CD40L-induced mDCs were susceptible to HIV-1 infection and mediated efficient viral transmission to CD4+ T cells. Although HIV-1 cis-infection was partially restricted in TNF-α-induced mDCs and profoundly blocked in LPS-induced mDCs, these cells efficiently promoted HIV-1 trans-infection of CD4+ T cells. The postentry restriction of HIV-1 infection in LPS-induced mDCs was identified at the levels of reverse transcription and postintegration, using real-time PCR quantification of viral DNA and integration. Furthermore, nucleofection of DCs with HIV-1 proviral DNA confirmed that impaired gene expression of LPS-induced mDCs was responsible for the postentry restriction of HIV-1 infection. Our results suggest that various DC subsets in vivo may differentially contribute to HIV-1 dissemination via dissociable cis- and trans-infections.
Dendritic cells (DCs), a group of professional antigen-presenting cells, play an important role in the induction and regulation of the adaptive immune response (5). Immature DCs (iDCs) located in submucosal tissues interact with pathogens and migrate to lymphoid tissues, where they become mature DCs (mDCs) and potently present antigens to T cells. DCs are among the first cells that encounter human immunodeficiency virus type 1 (HIV-1) at the mucosa, and they are thought to play an important role in the initial stages of HIV-1 infection and dissemination (for a review, see reference 52). DCs transmit HIV-1 to CD4+ T cells through trans- and cis-infection pathways. Trans-infection mediated by DCs can occur across infectious synapses (3, 20, 33, 46) and via exocytosis of HIV-associated exosomes (50). Cis-infection of DCs can lead to virus production and long-term transmission of HIV-1 (8, 19, 22, 23, 30, 37, 45, 46). It is conceivable that both of these mechanisms may coexist and contribute to viral dissemination. However, it is unclear whether DC-mediated HIV-1 trans- and cis-infections are associated or interdependent.
DCs can be infected by HIV-1 and support low levels of viral replication, suggesting that HIV-1-infected DCs may be a viral reservoir in vivo (52). DC maturation suppresses HIV-1 infection through multifaceted mechanisms, which involve decreased viral fusion (9), a block to reverse transcription (RT) (22, 23), and a postintegration restriction that has been proposed to exist at the transcriptional level (4). In contrast to the inefficient HIV-1 cis-infection of mDCs relative to that of iDCs, HIV-1 trans-infection of CD4+ T cells is enhanced by DC activation or maturation (9, 10, 14, 22, 33, 40, 48). Increased mDC-T-cell contact facilitates the formation of infectious synapses (20, 33, 40, 46, 48), which may partially account for enhanced HIV-1 trans-infection efficiency.
Various stimuli (40, 52), including lipopolysaccharides (LPS), tumor necrosis factor alpha (TNF-α), and the CD40 ligand (CD40L), have been used to induce effective DC maturation in vitro. LPS- and TNF-α-induced DC maturation may recapitulate inflammatory stimulation of DCs in vivo (44). The interactions between CD40L on T cells and its counterreceptor, CD40, on DCs are critical for antigen-specific T-cell responses (23, 34, 59). Thus, CD40L stimulation may represent the activation of DCs through T-cell interactions in vivo. Sanders et al. compared HIV-1 transmission efficiencies mediated by DCs that matured in response to different stimuli (40); however, the relative efficiencies of HIV-1 replication in various types of DCs remain to be defined.
Using replication-competent and single-cycle HIV-1, we compared cis- and trans-infections of HIV-1 mediated by monocyte-derived iDCs and various stimulus-induced mDCs. We found that DC-mediated cis- and trans-infections of HIV-1 are dissociable. Compared with the infection of iDCs and CD40L-induced mDCs, HIV-1 infection was partially restricted in TNF-α-induced mDCs and profoundly blocked in LPS-induced mDCs; however, these cells efficiently enhanced HIV-1 trans-infection of CD4+ T cells. Moreover, restricted RT and impaired gene expression contributed to the postentry block of HIV-1 infection in LPS-induced mDCs. Together, our results provide new insights into HIV-1 infection and dissemination mediated by DCs, suggesting that various DC subsets in vivo may facilitate HIV-1 spread via dissociable cis- and trans-infection pathways.
CD14+ monocytes in human peripheral blood mononuclear cells were separated from the buffy coats of healthy donors (provided by the Blood Center of Wisconsin, Milwaukee, WI) as previously described (47). Immature DCs were generated from purified monocytes treated with 50 ng/ml of granulocyte-macrophage colony-stimulating factor and interleukin 4 for 5 to 6 days as described previously (47). Immature DCs were >98.5% pure by DC-SIGN, HLA-DR, CD11b, and CD11c staining but were negative for CD3 and CD14. Various types of mDCs were differentiated by separately culturing iDCs with 10 ng/ml of LPS (Escherichia coli strain O55:B5; Sigma-Aldrich), 25 ng/ml of TNF-α, or 0.5 μg/ml of CD40L for an additional 2 days (all cytokines were purchased from PeproTech, Rocky Hill, NJ). Endotoxin levels of all recombinant cytokines were <0.1 ng/μg. The human embryonic kidney cell line HEK293T, CD4+ T-cell line Hut/CCR5, and HIV-1 indicator cell line GHOST/R5 (kind gifts from Vineet KewalRamani, National Cancer Institute, Frederick, MD) have been described (54).
DCs were stained with specific monoclonal antibodies (MAbs) or isotype-matched immunoglobulin G (IgG) controls as previously described (51). Phycoerythrin-conjugated mouse anti-human MAbs (BD Biosciences [unless specified otherwise]) against the following molecules and isotype-matched IgG control MAbs were used for staining: CD4 (clone S3.5; Caltag Laboratories), CD11c (clone BU15), CD14 (clone TÜK4), HLA-DR (clone TÜ 36), CD86 (clone 2331), CCR5 (clone 45531; R&D Systems), and CXCR4 (clone 44717; R&D Systems). Stained cells were analyzed using a FACSCalibur flow cytometer (Becton Dickinson). DC viability was examined with an annexin V-phycoerythrin apoptosis detection kit (BD Pharmingen), using flow cytometry.
Single-cycle luciferase reporter HIV-1 stocks were generated by cotransfection of HEK293T cells with pLai3ΔenvLuc2 (56), an env-deleted and nef-inactivated HIV-1 proviral construct (a kind gift from Michael Emerman, Fred Hutchinson Cancer Research Center, Seattle, WA), and an expression plasmid for vesicular stomatitis virus G protein (VSV-G) or R5 HIV-1 envelope JRFL, as previously described (47). The infectivities of the virus stocks were evaluated by limiting dilution on GHOST/R5 cells (54). Replication-competent HIV-1NLAD8 was generated by transfection of HEK293T cells with the proviral construct pNLAD8 (18) (a kind gift from Eric Freed, National Cancer Institute, Frederick, MD). To minimize the contamination of HIV-1NLAD8 stocks with plasmid DNA, Hut/CCR5 cells (1 × 106) were infected with HEK293T-derived HIV-1NLAD8 (5 ng of p24), and supernatants were harvested at 5 days postinfection (dpi). Gag p24 concentrations of HIV-1NLAD8 stocks were measured using an enzyme-linked immunosorbent assay (ELISA; anti-p24-coated plates were purchased from the AIDS Vaccine Program, SAIC, Frederick, MD).
To measure HIV-1 entry, various types of DCs (2 × 105) were pulsed separately with HIV-Luc/VSV-G (multiplicity of infection [MOI], 0.5) or HIV-1NLAD8 (20 ng of p24) for 2 h at 37°C, and cells were trypsinized after extensive washes and then lysed with 200 μl of 1% Triton X-100 buffer for Gag p24 quantification by ELISA as previously described (47). HIV-1 transmission and direct infection assays using luciferase reporter HIV-1 were performed as previously described (47, 53). Cell lysates were analyzed for luciferase activity with a commercially available kit (Promega). For infection of HIV-1NLAD8, DCs (2 × 105) were pulsed with HIV-1NLAD8 (20 ng of p24) at 37°C for 2 h. When indicated, DCs were pretreated with 1 μM of the HIV-1 fusion inhibitor T-20 (NIH AIDS Research and Reference Reagent Program) at 37°C for 30 min, and T-20 was maintained at the same concentration during viral incubation and infection.
DCs (2 × 105) were infected with HIV-Luc/VSV-G (MOI, 2.5) or HIV-1NLAD8 (20 ng of p24) that had been pretreated with 60 U/ml of DNase I (Fermenta) at 37°C for 1 h as previously described (29). At 2 dpi, total cellular DNA was extracted from the infected DCs with a blood DNA kit (QIAGEN). Cellular DNA was quantified with a spectrophotometer (NanoDrop), normalized, and confirmed by real-time PCR quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, using specific primers (forward, 5′-TGG ATA TTG CCA TCA ATG ACC-3′; reverse, 5′-GAT GGC ATG GAC TGT GGT CAT G-3′).
Primers, probes, and real-time PCR conditions for the detection of HIV-1 late RT products, one-long-terminal-repeat (1-LTR) circles, and 2-LTR circles were previously described (28, 29). The late RT products were amplified with an LTR R-specific primer (forward; 5′-GGG AGC TCT CTG GCT AAC T-3′) and a gag-specific primer (reverse; 5′-GGA TTA ACT GCG AAT CGT TC-3′). The primers used for the detection of 1-LTR circles were LA1 (forward; 5′-GCG CTT CAG CAA GCC GAG TCC T-3′) and LA15 (reverse; 5′-CAC ACC TCA GGT ACC TTT AAG A-3′). For 2-LTR circle detection, a U5-specific primer (forward; 5′-GCC TGG GAG CTC TCT GGC TAA-3′) and a U3-specific primer (reverse; 5′-GCC TTG TGT GTG GTA GAT CCA-3′) were used.
Integrated HIV-1 proviral DNA was quantified by real-time PCR as previously described, with slight modifications (55). The following primers were used: Alu forward (5′-AGC CTC CCG AGT AGC TGG GA-3′), Alu reverse (5′-TGC TGG GAT TAC AGG CGT GAG-3′), first-gag reverse (5′-CAA TAT CAT ACG CCG AGA GTG CGC G CTT CAG CAA G-3′, with the underlined artificial tag sequence to improve amplification specificity), second-LTR forward (5′-TTG TTA CAC CCT ATG AGC CAG C-3′), and second-tag reverse (5′-CAA TAT CAT ACG CCG AGA GTG C-3′). Fluorescence-labeled specific probes were 5′-(6-carboxyfluorescein)-TCG ACG CAG GAC TCG GCT TGC T-(6-carboxytetramethylrhodamine)-3′ for the detection of late RT products and 5′-(6-carboxyfluorescein)-AAG TAG TGT GTG CCC GTC TGT TGT GTG ACT C-(6-carboxytetramethylrhodamine)-3′ for the detection of 1-LTR circles, 2-LTR circles, and integrants. PCRs were performed using iQ Supermix kits (Bio-Rad); primers and probes (Integrated DNA Technologies, Coralville, IA) were used at final concentrations of 200 nM and 100 nM, respectively. A hot start was performed by heating the reaction mixtures for 10 min at 95°C. The cycling conditions were 45 cycles of 95°C for 15 s and 60°C for 1 min, using an iCycler real-time PCR system (Bio-Rad).
As standards for real-time PCR, serial dilutions (106 to 101 copies) of plasmid pNLAD8 were used for the late RT reactions, and a plasmid (pSK-CJ380) containing a 2-LTR circle (29) (a kind gift from Stephen Hughes, National Cancer Institute, Frederick, MD) was used for 2-LTR circle reactions. A plasmid (pCR-1-LTR) containing a 1-LTR circle was used as a standard for 1-LTR circle detection. The pCR-1-LTR plasmid was constructed by T-A cloning of 1-LTR PCR products from HIV-1NLAD8-infected Hut/CCR5 cells, using the pCR2.1 vector (Invitrogen), and was confirmed by sequencing. Cellular DNA extracted from Hut/CCR5 cells stably transduced with single-cycle HIV-1/VSV-G was used as a standard for HIV-1 integrants. The copy numbers of integrated proviral DNA (1.8 copies/cell) were confirmed by real-time PCR quantification of late RT products and GAPDH.
DCs (2 × 105) were infected with HIV-Luc/VSV-G (MOI, 2.0). At 2 dpi, total cellular RNA from infected cells was extracted with an RNeasy Mini kit (QIAGEN) and treated with an RNase-free DNase set (QIAGEN). The first-strand cDNA was synthesized using 200-ng RNA templates with a Superscript II first-strand synthesis kit and random hexamer primers (Invitrogen). Specific primers (Gag-F, 5′-CTA GAA CGA TTC GCA GTT AAT CCT-3′; and Gag-R, 5′-CTA TCC TTT GAT GCA CAC AAT AGA G-3′) were used to amplify a fragment of gag cDNA. Primers specific for GAPDH were used as an internal control. PCRs were performed with 50-μl reaction mixtures containing 5 μl of cDNA products as the template. After the initial incubation at 95°C for 3 min, 30 cycles were carried out (15 s at 95°C, followed by 30 s at 56°C and 10 s at 72°C), with a final 5-min incubation at 72°C.
DCs (0.8 × 106 to 1 × 106) were transfected separately with plasmid pmaxGFP (2 μg) or pNLAD8 (3 μg), using a Nucleofector device (Amaxa) with the U-002 program and a human DC-specific kit (Amaxa). The expression of green fluorescent protein (GFP) in pmaxGFP-transfected DCs was analyzed at 24 h posttransfection by flow cytometry. HIV-1 Gag p24 in the supernatants (from a total of 1 ml) and cell lysates (from a total of 400 μl) of pNLAD8-transfected DCs was quantified by ELISA at 5 days posttransfection. Cell-free supernatants (0.6 ml from a total of 1 ml) from pNLAD8-transfected DCs were used to infect GHOST/R5 cells (2 × 105). HIV-1 replication was measured by GFP expression at 3 dpi, using flow cytometry.
Statistical analyses were performed using Wilcoxon's paired t test with the Prism program.
Purified human CD14+ monocytes from healthy donors were differentiated into iDCs in vitro. Immature DCs were activated separately with LPS, TNF-α, and CD40L for 2 days to generate various types of mDCs (referred to as mDC-LPS, mDC-TNF-α, and mDC-CD40L, respectively). To confirm the phenotypic markers of iDCs and mDCs, DCs were immunostained and analyzed by flow cytometry. As expected, all of the DC subsets expressed high levels of CD11c (Fig. 1A and B), which is a characteristic marker of myeloid-derived DCs. Efficient DC maturation upregulates the cell surface expression of HLA-DR and CD86 (5). Compared with iDCs, the HLA-DR- and CD86-positive populations were largely increased in the different types of mDCs (Fig. (Fig.1A).1A). The relative expression levels of HLA-DR and CD86 were also increased in different types of mDCs compared with those in iDCs (Fig. (Fig.1B).1B). These results demonstrated that iDCs were effectively activated and differentiated into various types of mDCs by different stimuli.
To compare the expression of HIV-1 receptors among different types of DCs, surface expression levels of CD4, CCR5, and CXCR4 on DCs were measured by flow cytometry. Similar levels of CD4 expression were observed among various types of DCs (Fig. (Fig.1C).1C). Relative to those in iDCs, CD4 levels in mDC-TNF-α were slightly increased, but they were slightly decreased in mDC-LPS (Fig. (Fig.1C).1C). Low levels of CCR5 and CXCR4 expression (5 to 11% positive populations) were observed among various types of DCs on day 7 of differentiation (data not shown).
To overcome potential differences in HIV-1 entry into various types of DCs because of the different expression levels of viral receptors, a single-cycle HIV-1 pseudotyped with VSV-G (HIV-VSV-G) was used to infect the different types of DCs. The HIV-1 proviral genome (HIV-Luc) has env deleted and nef inactivated with a luciferase reporter insertion but contains all other viral genes (56). Various types of DCs were infected with increasing amounts of HIV-Luc/VSV-G for 2 h, and viral infection was determined at 4 dpi by measuring the luciferase activities of cell lysates.
HIV-1 infection of iDCs, mDC-TNF-α, and mDC-CD40L was enhanced with a fivefold increase of viral input (Fig. (Fig.2A).2A). At the same MOI, HIV-Luc/VSV-G infection in mDC-CD40L was more efficient than that in other types of DCs. At an MOI of 2.5, HIV-1 infection of mDC-CD40L was 1.7-fold and 4.2-fold more efficient than that of iDCs and mDC-TNF-α, respectively. In contrast, mDC-LPS did not support HIV-1 infection, even with larger amounts of virus and an extended time course (Fig. 2A and B). To examine whether HIV-1 infection in mDC-LPS was delayed, various types of DCs were infected with HIV-Luc/VSV-G at an MOI of 0.2 and infection was measured at 3, 5, and 7 dpi. Results obtained with HIV-1 infection of the various types of DCs indicated that HIV-1 infection is profoundly blocked in mDC-LPS (Fig. (Fig.2B).2B). No significant differences in DC viability were observed among the various types of DCs.
To compare the efficiencies of viral entry into various types of DCs, DCs were infected with HIV-Luc/VSV-G for 2 h at 37°C and trypsinized after extensive washes to strip cell surface-bound HIV-1, and the Gag p24 in DC lysates was measured. The p24 level in mDC-LPS was two- to threefold higher (P < 0.05) than those in other types of DCs (Fig. (Fig.2C),2C), indicating increased HIV-1 endocytosis in mDC-LPS. Comparable p24 levels were detected in iDCs, mDC-TNF-α, and mDC-CD40L (Fig. (Fig.2C).2C). Together, these data suggest a strong postentry block to HIV-1 replication in mDC-LPS.
To examine the restriction of the HIV-1 life cycle in mDC-LPS, HIV-1 DNA and integration were quantified. Cellular DNAs from HIV-Luc/VSV-G-infected DCs at 2 dpi were subjected to real-time PCR detection, using specific primers and Taqman probes for HIV-1 late RT products, LTR circles, and integrated viral DNA. Given that the HIV-1 stocks were generated by transfection of proviral DNA and to avoid plasmid DNA contamination in PCR detection, HIV-1 stocks were pretreated with DNase I before the infection. Diagnostic PCR analysis confirmed that the DNase I treatment completely removed plasmid DNA remaining in viral stocks (Fig. (Fig.3A3A).
Lower levels of late RT products from the infected DCs were obtained at 1 dpi than at 2 dpi (not shown), and therefore all viral DNA detections were performed at 2 dpi. Unexpectedly, three- to fourfold (P < 0.05) more HIV-1 late RT products were found in mDC-LPS than in other types of DCs (Fig. (Fig.3B),3B), which correlated with a two- to threefold enhanced HIV-1 uptake in mDC-LPS (Fig. (Fig.2C).2C). The late RT products were present at 23,700 copies per 50 ng of cellular DNA (equivalent to 63 cells/ng or 7.5 copies/cell) from infected mDC-LPS. Similar levels of late RT products (1.9 to 2.4 copies/cell) were detected in iDCs, mDC-TNF-α, and mDC-CD40L (Fig. (Fig.3B).3B). Thus, the postentry restriction of HIV-Luc/VSV-G infection in mDC-LPS was not caused by a block in RT.
To determine whether nuclear import of HIV-1 RT products might cause the postentry restriction in mDC-LPS, 1-LTR and 2-LTR circles were quantified in infected DCs. Although 1-LTR and 2-LTR circles produced from fully reverse-transcribed retroviral DNA are abortive products for integration, they can be used as surrogate markers for nuclear import of the viral DNA (17). The amounts of 1-LTR circles in iDCs, mDC-LPS, mDC-TNF-α, and mDC-CD40L were 702, 149, 222, and 2,610 copies (per 100 ng of cellular DNA), respectively. No detectable 2-LTR circles were found in mDC-LPS at 2 dpi, whereas 49 to 297 copies (per 250 ng of cellular DNA) of 2-LTR circles were detected in the other types of DCs (Fig. (Fig.3C).3C). These data suggest that the nuclear import of HIV-1 DNA might contribute to the restriction of HIV-1 infection in mDC-LPS. To test this hypothesis, the amount of proviral DNA in infected DCs was quantified using Alu-gag-based real-time PCR. Strikingly, HIV-1 integrant levels in HIV-Luc/VSV-G-infected mDC-LPS (equivalent to 0.4 copy/cell) were 2- to 4-fold higher than those in iDCs and mDC-TNF-α but 1.8-fold lower than that in mDC-CD40L (Fig. (Fig.3D).3D). Furthermore, RT-PCR detection of gag mRNA expression indicated that the transcription of viral mRNA was impaired by 60% to 70% in HIV-1-infected mDC-LPS and mDC-TNF-α relative to that in iDCs and mDC-CD40L (Fig. (Fig.3E).3E). Together, these data suggest that the postentry restriction of HIV-VSV-G in mDC-LPS is mainly due to a postintegration block.
To investigate whether wild-type HIV-1 infection in DCs is similar to that by HIV-VSV-G, DC infection with replication-competent HIV-1 was examined. Previous studies have shown that iDCs are more susceptible to R5-tropic HIV-1 than to X4-tropic HIV-1 (47, 52), and therefore the R5-tropic strain HIV-1NLAD8 (18) was used. To compare the efficiencies of viral entry among various types of DCs, DCs were first pulsed with HIV-1NLAD8 for 2 h at 37°C and then trypsinized after extensive washes to strip cell surface-bound HIV-1. The Gag p24 levels in the lysates of DCs were quantified. Given that HIV-1 can enter DCs via endocytosis and viral fusion, the HIV-1 fusion inhibitor T-20 was used to block fusion-mediated viral entry. T-20 is a small synthetic peptide, corresponding to a region of HIV-1 gp41, which potently inhibits fusion-mediated HIV-1 entry and cell-cell fusion (49).
In the absence of T-20, the p24 level in mDC-LPS was five- to eightfold (P < 0.01) higher than those in other DC types (Fig. (Fig.4A),4A), indicating enhanced HIV-1 uptake into mDC-LPS. Compared with that into iDCs, viral entry into mDC-TNF-α and mDC-CD40L was slightly decreased (Fig. (Fig.4A).4A). Blocking viral fusion with T-20 did not result in a decrease of viral entry into DCs. In fact, p24 levels in mDC-LPS and mDC-TNF-α were slightly increased (about 1.3-fold) in the presence of T-20 (Fig. (Fig.4A).4A). These results suggest that HIV-1NLAD8 entered these DCs predominately via endocytosis.
To examine HIV-1NLAD8 replication in DCs, Gag p24 levels in supernatants from virus-pulsed DCs were measured at 3, 5, and 7 dpi. T-20 was used to block viral fusion during the infection. In the absence of T-20, the amount of p24 in HIV-1NLAD8-infected cells increased five- to sixfold in iDCs, mDC-TNF-α, and mDC-CD40L from 3 dpi to 7 dpi, while low levels of p24 (330 to 410 pg/ml) were present in mDC-LPS supernatants during the infection (Fig. (Fig.4B).4B). Similar levels of p24 were detected in supernatants from infected iDCs and mDC-CD40L and were higher than that in supernatant from mDC-TNF-α. In the presence of T-20, viral replication in iDCs, mDC-TNF-α, and mDC-CD40L was efficiently blocked, indicating that HIV-1 replication in these cells requires fusion-mediated entry. In contrast, T-20 had no effect in mDC-LPS (Fig. (Fig.4B),4B), indicating fusion-independent viral endocytosis in mDC-LPS. Incubation of HIV-infected DCs with the reverse transcriptase inhibitor azidothymidine efficiently blocked the p24 in the supernatants (not shown), suggesting that the low levels of p24 released by mDC-LPS may result from a small amount of progeny viruses generated from endocytosed HIV-1.
To investigate restricted HIV-1 replication in mDC-LPS, HIV-1 DNA from HIV-1NLAD8 infected DCs was quantified at 2 dpi by real-time PCR. Basal levels of HIV-1 late RT products (24 copies/50 ng cellular DNA; detection limit, 10 copies) were found in mDC-LPS, while 40- to 80-fold (P < 0.0001) more late RT products were detected in other types of infected DCs (Fig. (Fig.4C).4C). As expected, 2-LTR circles were undetectable in infected mDC-LPS but were measured in other types of DCs, in the range of 232 to 482 copies per 250 ng cellular DNA (Fig. (Fig.4D).4D). These data indicate that restricted RT is responsible for the postuptake block of wild-type HIV-1 replication in mDC-LPS.
To examine whether endocytosed virus in DCs can produce viral DNA, various types of DCs were pulsed separately with HIV-1NLAD8 and cultured for 12 h before detection of late RT products. T-20 was used to block HIV-1 fusion during the infection. In the absence of T-20, HIV-1 late RT products were present at 751, 54, 101, and 836 copies per 50 ng of cellular DNA from iDCs, mDC-LPS, mDC-TNF-α, and mDC-CD40L, respectively (Fig. (Fig.5A).5A). In contrast, only basal levels of late RT products (14 to 27 copies) were detected in infected DCs in the presence of T-20 (Fig. (Fig.5A),5A), suggesting that fusion-mediated viral entry is required for productive synthesis of viral DNA.
As controls, the amounts of HIV-1 present in infected DCs were measured, which comprised both the retained input HIV-1 and newly synthesized viruses. Various types of DCs were pulsed separately with HIV-1NLAD8, cultured for 12 h, and then trypsinized before the cells were lysed for p24 detection. In the absence of T-20, the p24 level in mDC-LPS was six- to sevenfold (P < 0.01) higher than those in other types of DCs (Fig. (Fig.5B).5B). However, T-20 treatment failed to reduce p24 in various types of DCs, suggesting that endocytosis-mediated HIV-1 entry is the predominant pathway in DCs. The amount of HIV-1 p24 in mDC-LPS was 7- to 10-fold (P < 0.01) higher than those in other types of DCs. Similar levels of p24 were detected in iDCs, mDC-TNF-α, and mDC-CD40L (Fig. (Fig.5B).5B). Together, these results suggest that HIV-1 fusion-mediated entry into DCs results in productive RT and viral replication, while endocytosis-mediated entry does not lead to efficient viral DNA synthesis or replication.
To compare HIV-1 production and to avoid potential discrepancies in HIV-1 fusion, RT, and nuclear import among various types of DCs, nucleofection of HIV-1 proviral DNA was used to generate HIV-1 in DCs. Nucleofection has been used as an efficient technique to deliver genes to nondividing cells, including human DCs (7). Various types of DCs were subjected to nucleofection with the proviral construct pNLAD8, and Gag p24 in supernatants and cell pellets was measured at 5 days posttransfection. The p24 levels in mDC-LPS samples (≤13 pg/ml) were below the detectable limit, while p24 levels generated in other types of DCs ranged from 190 to 1,420 pg/ml in supernatants and 100 to 470 pg/ml in cell pellets (Fig. (Fig.6A).6A). Transfected mDC-TNF-α generated the highest levels of HIV-1 p24 in cells and supernatants (Fig. (Fig.6A),6A), suggesting that potential restrictions of the HIV-1 early life cycle may account for less viral replication in HIV-1NLAD8-infected mDC-TNF-α. To examine the transfection efficiency of mDC-LPS, real-time PCR quantification of pNLAD8 plasmid DNA from transfected iDCs and mDC-LPS was performed at 30 min postnucleofection. DCs incubated with pNLAD8 plasmid DNA but without nucleofection were used as background controls. The copy number of pNLAD8 DNA recovered from nucleofected mDC-LPS was 2.8-fold higher than that from iDCs, indicating that the lack of HIV-1 production from the nucleofected mDC-LPS was probably not due to a low efficiency of transfection.
To measure the infectivity of HIV-1 generated from pNLAD8-nucleofected DCs, GHOST/R5 cells were infected with the supernatants of the nucleofected DCs. GHOST/R5 cells are human osteosarcoma cells that express CD4 and CCR5. These cells contain a GFP gene under the control of the HIV-2 LTR promoter, which is expressed during HIV-1 infection via Tat transactivation acting as an indicator of infection (11). HIV-1 infection (GFP expression) in GHOST/R5 cells was measured using flow cytometry. Background levels of GFP-positive GHOST/R5 cells (0.61%) were observed when the cells were infected with the supernatants from nucleofected mDC-LPS, whereas 2% to 9% GFP-positive GHOST/R5 cells were detected when the cells were infected with the supernatants from other types of DCs (Fig. (Fig.6B).6B). Thus, transfection of HIV-1 proviral DNA can generate infectious HIV-1 in iDC, mDC-TNF-α, and mDC-CD40L but not in mDC-LPS, suggesting that there is a strong block to HIV-1 gene expression in mDC-LPS.
To investigate whether restricted HIV-1 gene expression in mDC-LPS was specific for the HIV-1 LTR promoter, a GFP reporter driven by a cytomegalovirus (CMV) promoter was used to examine exogenous gene expression in DCs. Various types of DCs were nucleofected with pmaxGFP, a GFP expression plasmid with a CMV promoter (a control for nucleofection). GFP expression was measured by flow cytometry at 24 h posttransfection. Only 6% GFP-positive mDC-LPS were detected, compared with 47%, 34%, and 37% GFP-positive iDCs, mDC-TNF-α, and mDC-CD40L, respectively (Fig. (Fig.6C).6C). When relative GFP expression levels were compared, mDC-LPS showed fourfold less GFP expression than did other types of DCs (Fig. (Fig.6D).6D). Consistent results were obtained from four independent experiments using DCs generated from different donors (not shown). Together, these results suggest that impaired gene expression in mDC-LPS contributes to the postentry restriction of HIV-1 replication.
To compare HIV-1 trans-infections of CD4+ T cells mediated by various types of DCs, viral transmission efficiency was examined using single-cycle and replication-competent HIV-1. After incubation with small amounts of HIV-1 for 2 h, DCs were washed and cocultured with a CD4+ T-cell line (Hut/CCR5 cells) for 2 days. Infection was determined by measuring the luciferase activities in cell lysates or Gag p24 levels in the supernatants at 2 dpi. Samples from cultures of DCs alone were used as controls for HIV-1 cis-infection.
Compared with controls with DC alone, trans-infection of HIV-1 pseudotyped with an R5 HIV-1 envelope (HIV-Luc/JRFL) in cocultures was enhanced 86-fold (P < 0.0001) by mDC-LPS, while iDCs, mDC-TNF-α, and mDC-CD40L mediated 15-, 26-, and 3-fold (P < 0.05) increases in viral transmission, respectively (Fig. (Fig.7A).7A). HIV-Luc/JRFL infection in mDC-LPS was strongly blocked (Fig. (Fig.7A7A and data not shown); however, upon coculture with CD4+ T cells, mDC-LPS were three- to fivefold more efficient than other types of DCs in the ability to transmit HIV-Luc/JRFL to CD4+ T cells (Fig. (Fig.7A).7A). Consistently, low levels of HIV-1 cis-infection were detected in mDC-CD40L without coculturing them with CD4+ T cells, which might help to explain the lower level of trans-infection mediated by mDC-CD40L (Fig. (Fig.7A7A).
Similar results were obtained using replication-competent HIV-1NLAD8. Compared with DC-only controls, HIV-1NLAD8 replication in cocultures was enhanced 4-, 171-, 62 (P < 0.0001)-, and 9-fold (P < 0.01) by iDCs, mDC-LPS, mDC-TNF-α, and mDC-CD40L, respectively (Fig. (Fig.7B).7B). Relative to iDCs, various types of mDCs appeared to transfer HIV-1 to CD4+ T cells more efficiently, despite the fact that there was no detectable viral replication in mDC-LPS and mDC-TNF-α at 2 dpi (Fig. (Fig.7B).7B). Low levels of HIV-1NLAD8 cis-infection were detected in iDCs and mDC-CD40L (Fig. (Fig.7B),7B), indicating that DC-mediated trans-infection is more efficient than cis-infection. Together, these results suggest that DCs potently mediate HIV-1 trans-infection, regardless of their ability to support viral replication.
Elucidating the mechanisms underlying DC-mediated HIV infection and viral transmission will enhance our understanding of HIV interactions with host cells (52). Here we provide evidence that cis- and trans-infections of HIV-1 mediated by DCs are dissociable pathways. HIV-1 cis-infection is restricted in mDC-LPS and impaired in mDC-TNF-α; however, these cells efficiently promoted HIV-1 trans-infection of CD4+ T cells, suggesting that, in vivo, the various DC subsets facilitate HIV-1 dissemination to different extents via cis- and trans-infections. It has been proposed that DCs transfer HIV-1 to CD4+ T cells in two phases (46). In the first phase (1 dpi), DC-captured HIV-1 is spread to CD4+ T cells via the trans-infection pathway. The second phase (2 to 3 dpi) occurs after de novo HIV-1 production by cis-infected DCs (46). However, our findings of dissociable cis- and trans-infections mediated by DCs suggest that these two processes are not necessarily sequential or interdependent.
Our results highlight the potential significance of DC-mediated HIV-1 transmission in vivo. In submucosal tissues, HIV-infected iDCs may act as a viral reservoir to further the spread of infection. HIV-1 coinfection with other sexually transmitted pathogens may induce DC maturation through inflammatory responses, such as upregulated cytokine production (43). Given their proximity to CD4+ T cells in lymphoid tissues, mDCs may potently stimulate HIV-1 transmission to these activated CD4+ T cells. A recent study indicated that significantly increased plasma LPS levels in HIV-infected humans correlate with systemic immune activation and AIDS progression (6). It is conceivable that increased LPS in HIV-infected individuals may induce DC maturation, thereby stimulating HIV-1 dissemination in vivo. Similarly, more recent reports indicated that LPS-activated CD34+ cell-derived Langerhans cells mediate efficient trans-infection of HIV-1 (10, 14). Despite the convenience of using monocyte-derived DCs, these cells may not fully mimic the physiological functions of the DC subsets that are involved in HIV infection in vivo. Thus, further studies are required to confirm these in vitro observations, using myeloid DCs, plasmacytoid DCs, Langerhans cells, and autologous CD4+ T cells from HIV-infected individuals.
DCs in vivo would likely be exposed to HIV-1 before or simultaneously to a maturation stimulus. It has been reported that HIV-infected blood myeloid DCs and monocyte-derived DCs fail to mature in culture (24, 25). Moreover, DCs from individuals with acute HIV infection have reduced expression of the costimulatory molecules CD80 and CD86, and this might influence DC-induced T-cell responses (31). These results suggest that productive HIV infection of DCs undermines the direct induction of T-cell-mediated immunity. In contrast, other studies have indicated that monocyte-derived DCs from HIV-infected individuals can efficiently induce cytotoxic T cells to respond to various antigens (15, 41). In addition, no functional defects in cytokine production were observed following stimulation of HIV-infected myeloid DCs and plasmacytoid DCs (45). In our study, to better compare HIV-1 infections among various types of DCs, viral infection was performed after generating mDCs from iDCs with different stimuli.
We found that distinct mechanisms contributed to postentry blocks of HIV-VSV-G and replication-competent HIV-1 in mDC-LPS. Thus, it is critical to consider the potential differences in using HIV-VSV-G instead of authentic HIV-1 to study postentry infection. Although endocytosis of both types of viruses was increased in mDC-LPS compared with that in other types of DCs, different viral trafficking pathways might affect the various steps in the viral life cycle. HIV-1-VSV-G enters cells through a low-pH-dependent endocytic pathway (1). Relative to those in other types of DCs, increased RT and efficient integration were detected in HIV-Luc/VSV-G-infected mDC-LPS, indicating that endocytosed HIV-VSV-G can complete uncoating, RT, and integration in mDC-LPS, while impaired gene expression in mDC-LPS could eventually block viral expression of the luciferase reporter in the infected cells. In contrast, in HIV-1NLAD8-infected mDC-LPS, restricted RT appeared to be an important component of the postentry block, and impaired gene expression in these cells might also play a role. For LPS-induced mDCs, it has been shown that HIV-1 is internalized into nonconventional, nonlysosomal, acidic compartments (20), which may restrict the RT process of endocytosed HIV-1NLAD8.
In addition to the restriction of RT, it has been proposed that there is a postentry block of HIV-1 in mDCs at the transcriptional level (4, 21). Using nucleofection of HIV-1 proviral DNA in DCs to overcome any restrictions in the early steps of the HIV-1 life cycle, we found that impaired gene expression in mDC-LPS contributed to the potent block of HIV-1 replication in these cells. Moreover, we observed that LPS-treated CD4+ T cells showed 1.2- to 1.4-fold increases in the efficiency of a single-cycle HIV-1 infection (not shown), suggesting that LPS does not generally induce restriction of HIV-1 infection in permissive cells. Consistent with this observation, an early study demonstrated that LPS is a potent stimulator of HIV-1 expression in monocytes and macrophages (39). The impaired HIV-1 gene expression in mDC-LPS might be due to inefficient transcription and/or translation. A comprehensive gene expression analysis showed that 225 transcripts among a total of 31,837 tag sequences were statistically different between iDCs and mDC-LPS (26). Moreover, treatment of iDCs with LPS activates several signal transduction pathways, including the NF-κB pathway (2). Therefore, the impaired gene expression in mDC-LPS is unlikely to be specific for HIV-1.
Although 2-LTR circles have been used as a marker for nuclear import of HIV-1 DNA, it is not always technically correct to interpret the lack of 2-LTR circles as an indicator of blocked nuclear import of viral DNA (57). Our data showed that 2-LTR circles were undetectable in HIV-VSV-G-infected mDC-LPS, while 1-LTR circles and integrated proviral DNA were detected in these cells. This might be due to the low abundance and degradation of 2-LTR circles in mDC-LPS. It has been reported that in HeLa-CD4 cells infected with HIV-1 at 2 dpi, most viral DNA is fully processed into integrated provirus (≈55%) and 1-LTR circles (≈35%), while only trace amounts of 2-LTR circles (<5%) can be detected (58). We estimated the abundance of LTR circles and integrants in various types of DCs infected with HIV-VSV-G. Interestingly, only 0.1 to 1% of the late RT products were converted into 2-LTR circles, while 0.3 to 22% and 4 to 35% of the late RT products were converted into 1-LTR circles and integrated proviral DNAs, respectively. These data suggest that the ratio of HIV-1 DNA to integrants might be cell type dependent.
The inefficient block of HIV-1 uptake into DCs by the fusion inhibitor T-20 suggests that HIV-1 entry into DCs occurs primarily through endocytosis rather than fusion. Nobile et al. reported that DC-SIGN, a C-type lectin molecule highly expressed on monocyte-derived DCs, facilitates HIV-1 capture and intracellular accumulation but inhibits viral fusion (36). This may help to explain the low levels of HIV-1 fusion and replication in DCs. We recently reported that mDC-LPS are more efficient than iDCs at transmitting HIV-1 to various types of target cells, independent of C-type lectins, including DC-SIGN (48). In addition, we observed that HIV-1 uptake by mDC-LPS was slightly increased (around 30%) by T-20 treatment for 2 to 12 h (Fig. (Fig.4A4A and and5B),5B), suggesting that there is a balance between HIV-1 fusion and endocytosis. The blockade of fusion-mediated HIV-1 entry by T-20 might decrease the degradation of Gag p24 in the cytosol, thereby indirectly increasing the amount of internalized HIV-1 in vesicles of mDC-LPS. A similar compensatory link between HIV-1 fusion and endocytosis has been reported for CD4+ primary T cells (42). In that study, blockade of CXCR4-tropic HIV virion fusion with AMD3100, a CXCR4-specific entry inhibitor, increased virion entry into CD4+ T cells via the endocytic pathway.
Although endocytosis-mediated HIV-1 entry can lead to productive viral infection in certain cell types, including human primary macrophages (13, 32), our results suggest that efficient RT and productive HIV-1 infection in DCs require fusion-mediated viral entry. A recent report indicated that HIV-1 enters Langerhans cells primarily by multiple-receptor-mediated endocytosis and that virions persist intact within the cytoplasm for several days without productive replication (27). The majority of endocytosed HIV-1 confined in intracellular compartments in DCs will eventually be degraded. In fact, rapid intracellular HIV-1 degradation has been reported for both iDCs and mDCs (35, 37, 46, 48).
CD40L-induced mDCs appeared to be more susceptible to HIV-VSV-G infection than were other types of DCs. This was likely due to more efficient nuclear import and integration of viral DNA in mDC-CD40L, as more 1-LTR and 2-LTR circles and proviruses were detected in the infected cells. In contrast, HIV-1NLAD8-infected mDC-CD40L and iDCs showed similar levels of viral replication, although viral uptake in mDC-CD40L was slightly lower than that in iDCs (Fig. 4A and B). These data suggest that the fusion of HIV-1NLAD8 is less efficient in mDC-CD40L. It has been shown that productive HIV-1 infection of DCs is triggered by CD40L treatment (16, 23); however, other studies have reported that HIV-1 infection is decreased in CD40L-induced mDCs (4, 34). These discrepant results might be due to different DCs and recombinant CD40L used in these studies.
Cellular restriction of HIV-1 infection in DCs may reflect innate antiviral immunity of the antigen-presenting cells. It has been reported that the antiretroviral factor APOBEC3G/3F (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G and 3F) mediates postentry resistance to HIV-1 infection in monocyte-derived iDCs (38). In addition, LPS-induced mDCs have reduced susceptibility to HIV-1 infection, which correlates with increased APOBEC3G levels (38). A recent report suggests that langerin-mediated HIV-1 internalization results in viral degradation, thus inhibiting HIV-1 replication in Langerhans cells (12). A better understanding of intrinsic antiretroviral immunity in DCs will provide new insights into more effective intervention strategies against HIV-1 infection and dissemination mediated by DCs.
We thank Eric Freed and Stephen Hughes for critical readings of the manuscript. We thank Michael Emerman, Eric Freed, Stephen Hughes, and Vineet KewalRamani for the kind gift of reagents. The fusion inhibitor T-20 was obtained from Roche through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
This work was supported by grants from the NIH (R01-AI068493) and the Campbell Foundation to L.W.
Published ahead of print on 8 August 2007.