Published Ahead of Print 8 August 2007. 2007, 81(20):11352. DOI: 10.1128/JVI.01081-07. J. Virol. 

J. Olson and Li WuChunsheng Dong, Alicia M. Janas, Jian-Hua Wang, Wendy 

-Infectiontrans- and cisReveals Dissociable in Immature and Mature Dendritic Cells

ReplicationImmunodeficiency Virus Type 1 Characterization of Human

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JOURNAL OF VIROLOGY, Oct. 2007, p. 11352–11362 Vol. 81, No. 200022-538X/07/$08.00�0 doi:10.1128/JVI.01081-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Human Immunodeficiency Virus Type 1 Replicationin Immature and Mature Dendritic Cells Reveals Dissociable

cis- and trans-Infection�

Chunsheng Dong, Alicia M. Janas, Jian-Hua Wang, Wendy J. Olson,† and Li Wu*Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road,

Milwaukee, Wisconsin 53226

Received 18 May 2007/Accepted 26 July 2007

Dendritic cells (DCs) transmit human immunodeficiency virus type 1 (HIV-1) to CD4� T cells through thetrans- and cis-infection pathways; however, little is known about the relative efficiencies of these pathways andwhether they are interdependent. Here we compare cis- and trans-infections of HIV-1 mediated by immatureDCs (iDCs) and mature DCs (mDCs), using replication-competent and single-cycle HIV-1. Monocyte-derivediDCs were differentiated into various types of mDCs by lipopolysaccharide (LPS), tumor necrosis factor alpha(TNF-�), and CD40 ligand (CD40L). iDCs and CD40L-induced mDCs were susceptible to HIV-1 infection andmediated efficient viral transmission to CD4� T cells. Although HIV-1 cis-infection was partially restricted inTNF-�-induced mDCs and profoundly blocked in LPS-induced mDCs, these cells efficiently promoted HIV-1trans-infection of CD4� T cells. The postentry restriction of HIV-1 infection in LPS-induced mDCs wasidentified at the levels of reverse transcription and postintegration, using real-time PCR quantification of viralDNA and integration. Furthermore, nucleofection of DCs with HIV-1 proviral DNA confirmed that impairedgene expression of LPS-induced mDCs was responsible for the postentry restriction of HIV-1 infection. Ourresults suggest that various DC subsets in vivo may differentially contribute to HIV-1 dissemination viadissociable cis- and trans-infections.

Dendritic cells (DCs), a group of professional antigen-pre-senting cells, play an important role in the induction and reg-ulation of the adaptive immune response (5). Immature DCs(iDCs) located in submucosal tissues interact with pathogensand migrate to lymphoid tissues, where they become matureDCs (mDCs) and potently present antigens to T cells. DCs areamong the first cells that encounter human immunodeficiencyvirus type 1 (HIV-1) at the mucosa, and they are thought toplay an important role in the initial stages of HIV-1 infectionand dissemination (for a review, see reference 52). DCs trans-mit HIV-1 to CD4� T cells through trans- and cis-infectionpathways. Trans-infection mediated by DCs can occur acrossinfectious synapses (3, 20, 33, 46) and via exocytosis of HIV-associated exosomes (50). Cis-infection of DCs can lead tovirus production and long-term transmission of HIV-1 (8, 19,22, 23, 30, 37, 45, 46). It is conceivable that both of thesemechanisms may coexist and contribute to viral dissemination.However, it is unclear whether DC-mediated HIV-1 trans- andcis-infections are associated or interdependent.

DCs can be infected by HIV-1 and support low levels of viralreplication, suggesting that HIV-1-infected DCs may be a viralreservoir in vivo (52). DC maturation suppresses HIV-1 infec-tion through multifaceted mechanisms, which involve de-creased viral fusion (9), a block to reverse transcription (RT)

(22, 23), and a postintegration restriction that has been pro-posed to exist at the transcriptional level (4). In contrast to theinefficient HIV-1 cis-infection of mDCs relative to that ofiDCs, HIV-1 trans-infection of CD4� T cells is enhanced byDC activation or maturation (9, 10, 14, 22, 33, 40, 48). In-creased mDC–T-cell contact facilitates the formation of infec-tious synapses (20, 33, 40, 46, 48), which may partially accountfor enhanced HIV-1 trans-infection efficiency.

Various stimuli (40, 52), including lipopolysaccharides(LPS), tumor necrosis factor alpha (TNF-�), and the CD40ligand (CD40L), have been used to induce effective DC mat-uration in vitro. LPS- and TNF-�-induced DC maturation mayrecapitulate inflammatory stimulation of DCs in vivo (44). Theinteractions between CD40L on T cells and its counterrecep-tor, CD40, on DCs are critical for antigen-specific T-cell re-sponses (23, 34, 59). Thus, CD40L stimulation may representthe activation of DCs through T-cell interactions in vivo. Sand-ers et al. compared HIV-1 transmission efficiencies mediatedby DCs that matured in response to different stimuli (40);however, the relative efficiencies of HIV-1 replication in vari-ous types of DCs remain to be defined.

Using replication-competent and single-cycle HIV-1, wecompared cis- and trans-infections of HIV-1 mediated bymonocyte-derived iDCs and various stimulus-induced mDCs.We found that DC-mediated cis- and trans-infections of HIV-1are dissociable. Compared with the infection of iDCs andCD40L-induced mDCs, HIV-1 infection was partially re-stricted in TNF-�-induced mDCs and profoundly blocked inLPS-induced mDCs; however, these cells efficiently enhancedHIV-1 trans-infection of CD4� T cells. Moreover, restrictedRT and impaired gene expression contributed to the postentryblock of HIV-1 infection in LPS-induced mDCs. Together, our

* Corresponding author. Mailing address: Department of Microbi-ology and Molecular Genetics, Medical College of Wisconsin, 8701Watertown Plank Road, Milwaukee, WI 53226. Phone: (414) 456-4075. Fax: (414) 456-6535. E-mail: liwu@mcw.edu.

† Present address: University of Wisconsin-Milwaukee, Milwaukee,WI 53201.

� Published ahead of print on 8 August 2007.

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results provide new insights into HIV-1 infection and dissem-ination mediated by DCs, suggesting that various DC subsetsin vivo may facilitate HIV-1 spread via dissociable cis- andtrans-infection pathways.

MATERIALS AND METHODS

Cell culture. CD14� monocytes in human peripheral blood mononuclear cellswere separated from the buffy coats of healthy donors (provided by the BloodCenter of Wisconsin, Milwaukee, WI) as previously described (47). ImmatureDCs were generated from purified monocytes treated with 50 ng/ml of granulo-cyte-macrophage colony-stimulating factor and interleukin 4 for 5 to 6 days asdescribed 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 10ng/ml of LPS (Escherichia coli strain O55:B5; Sigma-Aldrich), 25 ng/ml ofTNF-�, or 0.5 �g/ml of CD40L for an additional 2 days (all cytokines werepurchased from PeproTech, Rocky Hill, NJ). Endotoxin levels of all recombinantcytokines were �0.1 ng/�g. The human embryonic kidney cell line HEK293T,CD4� T-cell line Hut/CCR5, and HIV-1 indicator cell line GHOST/R5 (kindgifts from Vineet KewalRamani, National Cancer Institute, Frederick, MD) havebeen described (54).

Flow cytometry and antibodies. DCs were stained with specific monoclonalantibodies (MAbs) or isotype-matched immunoglobulin G (IgG) controls aspreviously described (51). Phycoerythrin-conjugated mouse anti-human MAbs(BD Biosciences [unless specified otherwise]) against the following moleculesand isotype-matched IgG control MAbs were used for staining: CD4 (clone S3.5;Caltag Laboratories), CD11c (clone BU15), CD14 (clone TUK4), HLA-DR(clone TU 36), CD86 (clone 2331), CCR5 (clone 45531; R&D Systems),and CXCR4 (clone 44717; R&D Systems). Stained cells were analyzed using aFACSCalibur flow cytometer (Becton Dickinson). DC viability was examinedwith an annexin V-phycoerythrin apoptosis detection kit (BD Pharmingen),using flow cytometry.

HIV-1 stocks. Single-cycle luciferase reporter HIV-1 stocks were generated bycotransfection of HEK293T cells with pLai3�envLuc2 (56), an env-deleted andnef-inactivated HIV-1 proviral construct (a kind gift from Michael Emerman,Fred Hutchinson Cancer Research Center, Seattle, WA), and an expressionplasmid for vesicular stomatitis virus G protein (VSV-G) or R5 HIV-1 envelopeJRFL, as previously described (47). The infectivities of the virus stocks wereevaluated by limiting dilution on GHOST/R5 cells (54). Replication-competentHIV-1NLAD8 was generated by transfection of HEK293T cells with the proviralconstruct pNLAD8 (18) (a kind gift from Eric Freed, National Cancer Institute,Frederick, MD). To minimize the contamination of HIV-1NLAD8 stocks withplasmid DNA, Hut/CCR5 cells (1 � 106) were infected with HEK293T-derivedHIV-1NLAD8 (5 ng of p24), and supernatants were harvested at 5 days postin-fection (dpi). Gag p24 concentrations of HIV-1NLAD8 stocks were measuredusing an enzyme-linked immunosorbent assay (ELISA; anti-p24-coated plateswere purchased from the AIDS Vaccine Program, SAIC, Frederick, MD).

HIV-1 entry, infection, and transmission assays. 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 at37°C, and cells were trypsinized after extensive washes and then lysed with 200�l of 1% Triton X-100 buffer for Gag p24 quantification by ELISA as previouslydescribed (47). HIV-1 transmission and direct infection assays using luciferasereporter HIV-1 were performed as previously described (47, 53). Cell lysateswere analyzed for luciferase activity with a commercially available kit (Promega).For infection of HIV-1NLAD8, DCs (2 � 105) were pulsed with HIV-1NLAD8 (20ng of p24) at 37°C for 2 h. When indicated, DCs were pretreated with 1 �M ofthe HIV-1 fusion inhibitor T-20 (NIH AIDS Research and Reference ReagentProgram) at 37°C for 30 min, and T-20 was maintained at the same concentrationduring viral incubation and infection.

Real-time PCR quantification of HIV-1 DNA and integration in infected DCs.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) at37°C for 1 h as previously described (29). At 2 dpi, total cellular DNA wasextracted from the infected DCs with a blood DNA kit (QIAGEN). CellularDNA was quantified with a spectrophotometer (NanoDrop), normalized, andconfirmed by real-time PCR quantification of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) DNA, using specific primers (forward, 5�-TGGATA TTG CCA TCA ATG ACC-3�; reverse, 5�-GAT GGC ATG GAC TGTGGT 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 werepreviously described (28, 29). The late RT products were amplified with an LTRR-specific primer (forward; 5�-GGG AGC TCT CTG GCT AAC T-3�) and agag-specific primer (reverse; 5�-GGA TTA ACT GCG AAT CGT TC-3�). Theprimers used for the detection of 1-LTR circles were LA1 (forward; 5�-GCGCTT CAG CAA GCC GAG TCC T-3�) and LA15 (reverse; 5�-CAC ACC TCAGGT 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-specificprimer (reverse; 5�-GCC TTG TGT GTG GTA GAT CCA-3�) were used.

Integrated HIV-1 proviral DNA was quantified by real-time PCR as previouslydescribed, with slight modifications (55). The following primers were used: Aluforward (5�-AGC CTC CCG AGT AGC TGG GA-3�), Alu reverse (5�-TGCTGG GAT TAC AGG CGT GAG-3�), first-gag reverse (5�-CAA TAT CATACG CCG AGA GTG CGC G CTT CAG CAA G-3�, with the underlinedartificial 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 specificprobes were 5�-(6-carboxyfluorescein)-TCG ACG CAG GAC TCG GCT TGCT-(6-carboxytetramethylrhodamine)-3� for the detection of late RT products and5�-(6-carboxyfluorescein)-AAG TAG TGT GTG CCC GTC TGT TGT GTGACT 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 startwas performed by heating the reaction mixtures for 10 min at 95°C. The cyclingconditions were 45 cycles of 95°C for 15 s and 60°C for 1 min, using an iCyclerreal-time PCR system (Bio-Rad).

As standards for real-time PCR, serial dilutions (106 to 101 copies) of plasmidpNLAD8 were used for the late RT reactions, and a plasmid (pSK-CJ380)containing a 2-LTR circle (29) (a kind gift from Stephen Hughes, NationalCancer 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 circledetection. The pCR-1-LTR plasmid was constructed by T-A cloning of 1-LTRPCR products from HIV-1NLAD8-infected Hut/CCR5 cells, using the pCR2.1vector (Invitrogen), and was confirmed by sequencing. Cellular DNA extractedfrom Hut/CCR5 cells stably transduced with single-cycle HIV-1/VSV-G was usedas a standard for HIV-1 integrants. The copy numbers of integrated proviralDNA (1.8 copies/cell) were confirmed by real-time PCR quantification of lateRT products and GAPDH.

RT-PCR detection of gag mRNA. DCs (2 � 105) were infected with HIV-Luc/VSV-G (MOI, 2.0). At 2 dpi, total cellular RNA from infected cells was extractedwith an RNeasy Mini kit (QIAGEN) and treated with an RNase-free DNase set(QIAGEN). The first-strand cDNA was synthesized using 200-ng RNA tem-plates with a Superscript II first-strand synthesis kit and random hexamer prim-ers (Invitrogen). Specific primers (Gag-F, 5�-CTA GAA CGA TTC GCA GTTAAT 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 wereused as an internal control. PCRs were performed with 50-�l reaction mixturescontaining 5 �l of cDNA products as the template. After the initial incubation at95°C for 3 min, 30 cycles were carried out (15 s at 95°C, followed by 30 s at 56°Cand 10 s at 72°C), with a final 5-min incubation at 72°C.

Nucleofection of DCs and infection of GHOST/R5 cells. DCs (0.8 � 106 to1 � 106) were transfected separately with plasmid pmaxGFP (2 �g) orpNLAD8 (3 �g), using a Nucleofector device (Amaxa) with the U-002 pro-gram and a human DC-specific kit (Amaxa). The expression of green fluo-rescent protein (GFP) in pmaxGFP-transfected DCs was analyzed at 24 hposttransfection by flow cytometry. HIV-1 Gag p24 in the supernatants (froma total of 1 ml) and cell lysates (from a total of 400 �l) of pNLAD8-transfected DCs was quantified by ELISA at 5 days posttransfection. Cell-freesupernatants (0.6 ml from a total of 1 ml) from pNLAD8-transfected DCswere used to infect GHOST/R5 cells (2 � 105). HIV-1 replication wasmeasured by GFP expression at 3 dpi, using flow cytometry.

Statistical analyses. Statistical analyses were performed using Wilcoxon’spaired t test with the Prism program.

RESULTS

Phenotypic cell surface markers of iDCs and mDCs. Puri-fied human CD14� monocytes from healthy donors were dif-ferentiated into iDCs in vitro. Immature DCs were activatedseparately with LPS, TNF-�, and CD40L for 2 days to generate

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various types of mDCs (referred to as mDC-LPS, mDC-TNF-�, and mDC-CD40L, respectively). To confirm the phe-notypic markers of iDCs and mDCs, DCs were immunostainedand analyzed by flow cytometry. As expected, all of the DCsubsets expressed high levels of CD11c (Fig. 1A and B), whichis a characteristic marker of myeloid-derived DCs. EfficientDC maturation upregulates the cell surface expression ofHLA-DR and CD86 (5). Compared with iDCs, the HLA-DR-and CD86-positive populations were largely increased in thedifferent types of mDCs (Fig. 1A). The relative expressionlevels of HLA-DR and CD86 were also increased in differenttypes of mDCs compared with those in iDCs (Fig. 1B). Theseresults demonstrated that iDCs were effectively activated anddifferentiated into various types of mDCs by different stimuli.

To compare the expression of HIV-1 receptors among dif-ferent types of DCs, surface expression levels of CD4, CCR5,and CXCR4 on DCs were measured by flow cytometry. Similarlevels of CD4 expression were observed among various types ofDCs (Fig. 1C). Relative to those in iDCs, CD4 levels in mDC-TNF-� were slightly increased, but they were slightly decreasedin mDC-LPS (Fig. 1C). Low levels of CCR5 and CXCR4expression (5 to 11% positive populations) were observedamong various types of DCs on day 7 of differentiation (datanot shown).

Comparison of VSV-G-pseudotyped HIV-1 infections amongvarious types of DCs reveals a postentry restriction in LPS-induced mDCs. To overcome potential differences in HIV-1entry into various types of DCs because of the different ex-pression levels of viral receptors, a single-cycle HIV-1pseudotyped with VSV-G (HIV-VSV-G) was used to infectthe different types of DCs. The HIV-1 proviral genome (HIV-Luc) has env deleted and nef inactivated with a luciferasereporter insertion but contains all other viral genes (56). Var-ious types of DCs were infected with increasing amounts ofHIV-Luc/VSV-G for 2 h, and viral infection was determined at4 dpi by measuring the luciferase activities of cell lysates.

HIV-1 infection of iDCs, mDC-TNF-�, and mDC-CD40Lwas enhanced with a fivefold increase of viral input (Fig. 2A).At the same MOI, HIV-Luc/VSV-G infection in mDC-CD40Lwas more efficient than that in other types of DCs. At an MOIof 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-�, respec-tively. 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 inmDC-LPS was delayed, various types of DCs were infectedwith HIV-Luc/VSV-G at an MOI of 0.2 and infection wasmeasured at 3, 5, and 7 dpi. Results obtained with HIV-1

FIG. 1. Cell surface markers of iDCs and mDCs. (A) Phenotypic surface markers of iDCs and mDCs. Monocyte-derived iDCs were separatelyinduced into different types of mDCs by treatment with LPS, TNF-�, or CD40L for 2 days. DC surface CD11c, HLA-DR, and CD86 were stainedwith specific MAbs or isotype-matched IgGs and analyzed by flow cytometry. (B) Relative expression levels of surface markers on iDCs and mDCs.All data show means standard deviations for duplicate samples. Data for one representative experiment out of three are shown. (C) Surface CD4expression on various types of DCs. Staining of DCs was done with either a murine IgG2a isotypic control antibody (thin lines) or CD4 MAb (thicklines). The mean fluorescence intensity of CD4 staining is shown in the top right corner of each histogram.

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infection of the various types of DCs indicated that HIV-1infection is profoundly blocked in mDC-LPS (Fig. 2B). Nosignificant differences in DC viability were observed among thevarious types of DCs.

To compare the efficiencies of viral entry into various typesof DCs, DCs were infected with HIV-Luc/VSV-G for 2 h at37°C and trypsinized after extensive washes to strip cell sur-face-bound HIV-1, and the Gag p24 in DC lysates was mea-sured. The p24 level in mDC-LPS was two- to threefold higher(P � 0.05) than those in other types of DCs (Fig. 2C), indi-cating increased HIV-1 endocytosis in mDC-LPS. Comparablep24 levels were detected in iDCs, mDC-TNF-�, and mDC-CD40L (Fig. 2C). Together, these data suggest a strongpostentry block to HIV-1 replication in mDC-LPS.

Postintegration restriction of VSV-G-pseudotyped HIV-1 inLPS-induced mDCs. To examine the restriction of the HIV-1life cycle in mDC-LPS, HIV-1 DNA and integration werequantified. Cellular DNAs from HIV-Luc/VSV-G-infectedDCs at 2 dpi were subjected to real-time PCR detection, usingspecific primers and Taqman probes for HIV-1 late RT prod-ucts, LTR circles, and integrated viral DNA. Given that theHIV-1 stocks were generated by transfection of proviral DNAand to avoid plasmid DNA contamination in PCR detection,HIV-1 stocks were pretreated with DNase I before the infec-tion. Diagnostic PCR analysis confirmed that the DNase Itreatment completely removed plasmid DNA remaining in vi-ral stocks (Fig. 3A).

Lower levels of late RT products from the infected DCswere obtained at 1 dpi than at 2 dpi (not shown), and thereforeall viral DNA detections were performed at 2 dpi. Unexpect-edly, three- to fourfold (P � 0.05) more HIV-1 late RT prod-ucts were found in mDC-LPS than in other types of DCs (Fig.3B), which correlated with a two- to threefold enhanced HIV-1uptake in mDC-LPS (Fig. 2C). The late RT products werepresent at 23,700 copies per 50 ng of cellular DNA (equivalentto 63 cells/ng or 7.5 copies/cell) from infected mDC-LPS. Sim-ilar levels of late RT products (1.9 to 2.4 copies/cell) weredetected in iDCs, mDC-TNF-�, and mDC-CD40L (Fig. 3B).Thus, the postentry restriction of HIV-Luc/VSV-G infection inmDC-LPS was not caused by a block in RT.

To determine whether nuclear import of HIV-1 RT prod-ucts might cause the postentry restriction in mDC-LPS, 1-LTRand 2-LTR circles were quantified in infected DCs. Although1-LTR and 2-LTR circles produced from fully reverse-tran-scribed retroviral DNA are abortive products for integration,they can be used as surrogate markers for nuclear import of theviral DNA (17). The amounts of 1-LTR circles in iDCs, mDC-LPS, mDC-TNF-�, and mDC-CD40L were 702, 149, 222, and2,610 copies (per 100 ng of cellular DNA), respectively. Nodetectable 2-LTR circles were found in mDC-LPS at 2 dpi,whereas 49 to 297 copies (per 250 ng of cellular DNA) of2-LTR circles were detected in the other types of DCs (Fig.3C). These data suggest that the nuclear import of HIV-1DNA might contribute to the restriction of HIV-1 infection inmDC-LPS. To test this hypothesis, the amount of proviralDNA 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- to4-fold higher than those in iDCs and mDC-TNF-� but 1.8-foldlower than that in mDC-CD40L (Fig. 3D). Furthermore, RT-PCR detection of gag mRNA expression indicated that thetranscription of viral mRNA was impaired by 60% to 70% inHIV-1-infected mDC-LPS and mDC-TNF-� relative to that iniDCs and mDC-CD40L (Fig. 3E). Together, these data suggestthat the postentry restriction of HIV-VSV-G in mDC-LPS ismainly due to a postintegration block.

RT of replication-competent HIV-1 is restricted in LPS-induced mDCs. To investigate whether wild-type HIV-1 infec-tion in DCs is similar to that by HIV-VSV-G, DC infectionwith replication-competent HIV-1 was examined. Previousstudies have shown that iDCs are more susceptible to R5-tropic HIV-1 than to X4-tropic HIV-1 (47, 52), and thereforethe R5-tropic strain HIV-1NLAD8 (18) was used. To comparethe efficiencies of viral entry among various types of DCs, DCs

FIG. 2. Comparison of VSV-G-pseudotyped HIV-1 infectionsamong various types of DCs reveals a postentry restriction in LPS-induced mDCs. (A and B) HIV-1 infection in mDC-LPS is blocked.(A) Various types of DCs (2 � 105) were separately infected withincreasing MOIs of VSV-G-pseudotyped, single-cycle luciferase re-porter HIV-1 (HIV-Luc/VSV-G). DCs were lysed at 4 dpi to deter-mine viral infection by measuring the luciferase activities of cell lysates.(B) DCs were infected with HIV-Luc/VSV-G at an MOI of 0.2, andinfection was determined at 3, 5, and 7 dpi. The luciferase activities ofthe infected mDC-LPS were at the levels (�100) of the mock infectioncontrols (not shown in panels A and B). cps, counts per second.(C) Enhanced entry of HIV-Luc/VSV-G into mDC-LPS. DCs werepulsed with HIV-Luc/VSV-G at an MOI of 0.5 at 37°C for 2 h, washed,trypsinized, and then lysed for Gag p24 detection. All data show themeans standard deviations for triplicate samples. Data for onerepresentative experiment out of four are shown.

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were first pulsed with HIV-1NLAD8 for 2 h at 37°C and thentrypsinized after extensive washes to strip cell surface-boundHIV-1. The Gag p24 levels in the lysates of DCs were quan-tified. Given that HIV-1 can enter DCs via endocytosis andviral fusion, the HIV-1 fusion inhibitor T-20 was used to blockfusion-mediated viral entry. T-20 is a small synthetic peptide,corresponding to a region of HIV-1 gp41, which potently in-hibits 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. 4A), indicating enhanced HIV-1 uptake into mDC-LPS.Compared with that into iDCs, viral entry into mDC-TNF-�and mDC-CD40L was slightly decreased (Fig. 4A). Blockingviral fusion with T-20 did not result in a decrease of viral entryinto DCs. In fact, p24 levels in mDC-LPS and mDC-TNF-�were slightly increased (about 1.3-fold) in the presence of T-20(Fig. 4A). These results suggest that HIV-1NLAD8 enteredthese DCs predominately via endocytosis.

To examine HIV-1NLAD8 replication in DCs, Gag p24 levelsin supernatants from virus-pulsed DCs were measured at 3, 5,and 7 dpi. T-20 was used to block viral fusion during theinfection. In the absence of T-20, the amount of p24 in HIV-1NLAD8-infected cells increased five- to sixfold in iDCs, mDC-TNF-�, and mDC-CD40L from 3 dpi to 7 dpi, while low levelsof p24 (330 to 410 pg/ml) were present in mDC-LPS superna-tants during the infection (Fig. 4B). Similar levels of p24 weredetected in supernatants from infected iDCs and mDC-CD40Land were higher than that in supernatant from mDC-TNF-�.

In the presence of T-20, viral replication in iDCs, mDC-TNF-�, and mDC-CD40L was efficiently blocked, indicatingthat HIV-1 replication in these cells requires fusion-mediatedentry. In contrast, T-20 had no effect in mDC-LPS (Fig. 4B),indicating fusion-independent viral endocytosis in mDC-LPS.Incubation of HIV-infected DCs with the reverse transcriptaseinhibitor azidothymidine efficiently blocked the p24 in the su-pernatants (not shown), suggesting that the low levels of p24released by mDC-LPS may result from a small amount ofprogeny 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 at2 dpi by real-time PCR. Basal levels of HIV-1 late RT products(24 copies/50 ng cellular DNA; detection limit, 10 copies) werefound in mDC-LPS, while 40- to 80-fold (P � 0.0001) morelate RT products were detected in other types of infected DCs(Fig. 4C). As expected, 2-LTR circles were undetectable ininfected mDC-LPS but were measured in other types ofDCs, in the range of 232 to 482 copies per 250 ng cellularDNA (Fig. 4D). These data indicate that restricted RT isresponsible for the postuptake block of wild-type HIV-1replication in mDC-LPS.

Endocytosed replication-competent HIV-1 in DCs does notproduce significant amounts of late RT products. To examinewhether 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

FIG. 3. Postintegration restriction of VSV-G-pseudotyped HIV-1 in LPS-induced mDCs. (A) DNase I treatment completely removed plasmidDNA in HIV stocks. HIV-Luc/VSV-G stocks were treated with DNase I (lane 2) or mock treated (lane 1) for 1 h at 37°C, inactivated with 1%Triton X-100, and then subjected to PCR detection using specific primers for the R-gag region of HIV proviral DNA. Lane M, 100-bp ladder; lane3, a negative control PCR using H2O as a DNA template. The arrow points to a band of specific PCR products (448 bp). (B to D) HIV-1 viralDNA quantification. DCs (2 � 105) were infected with HIV-Luc/VSV-G at an MOI of 2.5, and cellular DNAs were extracted at 2 dpi and subjectedto real-time PCR quantification of HIV-1 late RT products (B), 2-LTR circles (C), and integrated proviral DNAs (integrants) (D). All data showmeans standard deviations for triplicate or duplicate samples. Data for one representative experiment out of three are shown. (E) HIV-1 gagmRNA expression in infected DCs. DCs (2 � 105) were infected with HIV-Luc/VSV-G at an MOI of 2.5, and cellular RNAs were extracted at2 dpi and subjected to RT-PCR detection of gag mRNA. RT-PCR was performed with (�) and without () reverse transcriptase (RT). Theamplification of GAPDH was used as an internal control for RT-PCR. PCR products were analyzed by 1% agarose gel electrophoresis withethidium bromide staining (A and E).

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the absence of T-20, HIV-1 late RT products were present at751, 54, 101, and 836 copies per 50 ng of cellular DNA fromiDCs, mDC-LPS, mDC-TNF-�, and mDC-CD40L, respec-tively (Fig. 5A). In contrast, only basal levels of late RT prod-ucts (14 to 27 copies) were detected in infected DCs in thepresence of T-20 (Fig. 5A), suggesting that fusion-mediatedviral entry is required for productive synthesis of viral DNA.

As controls, the amounts of HIV-1 present in infected DCswere measured, which comprised both the retained inputHIV-1 and newly synthesized viruses. Various types of DCswere pulsed separately with HIV-1NLAD8, cultured for 12 h,and then trypsinized before the cells were lysed for p24 detec-tion. 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. 5B). However, T-20 treatment failed to reduce p24 invarious types of DCs, suggesting that endocytosis-mediatedHIV-1 entry is the predominant pathway in DCs. The amountof HIV-1 p24 in mDC-LPS was 7- to 10-fold (P � 0.01) higherthan those in other types of DCs. Similar levels of p24 weredetected in iDCs, mDC-TNF-�, and mDC-CD40L (Fig. 5B).Together, these results suggest that HIV-1 fusion-mediatedentry into DCs results in productive RT and viral replication,while endocytosis-mediated entry does not lead to efficientviral DNA synthesis or replication.

Impaired gene expression in mDC-LPS contributes to therestriction of HIV-1 replication. To compare HIV-1 produc-tion and to avoid potential discrepancies in HIV-1 fusion, RT,and nuclear import among various types of DCs, nucleofectionof HIV-1 proviral DNA was used to generate HIV-1 in DCs.Nucleofection has been used as an efficient technique to de-liver genes to nondividing cells, including human DCs (7).Various types of DCs were subjected to nucleofection with theproviral construct pNLAD8, and Gag p24 in supernatants andcell pellets was measured at 5 days posttransfection. The p24levels in mDC-LPS samples (�13 pg/ml) were below the de-tectable limit, while p24 levels generated in other types of DCsranged from 190 to 1,420 pg/ml in supernatants and 100 to 470pg/ml in cell pellets (Fig. 6A). Transfected mDC-TNF-� gen-erated the highest levels of HIV-1 p24 in cells and superna-tants (Fig. 6A), suggesting that potential restrictions of theHIV-1 early life cycle may account for less viral replication inHIV-1NLAD8-infected mDC-TNF-�. To examine the transfec-tion efficiency of mDC-LPS, real-time PCR quantification ofpNLAD8 plasmid DNA from transfected iDCs and mDC-LPSwas performed at 30 min postnucleofection. DCs incubatedwith pNLAD8 plasmid DNA but without nucleofection wereused as background controls. The copy number of pNLAD8DNA recovered from nucleofected mDC-LPS was 2.8-fold

FIG. 4. RT of replication-competent HIV-1 is restricted in LPS-induced mDCs. (A) HIV-1NLAD8 entry is enhanced in mDC-LPS. DC-associated Gag p24 was measured after incubation with HIV-1NLAD8 for 2 h at 37°C in the presence or absence of the HIV-1 fusion inhibitor T-20.After extensive washes, HIV-1-pulsed DCs were trypsinized and lysed for Gag p24 detection. (B) Restricted HIV-1 replication in mDC-LPS. DCswere infected with HIV-1NLAD8 in the presence or absence of T-20. Supernatants of infected DCs were measured for p24 levels at 3, 5, and 7 dpi.(C) Basal levels of HIV-1 late RT products in mDC-LPS. (D) Undetectable 2-LTR circles in mDC-LPS. Cellular DNAs of HIV-1NLAD8-infectedDCs were extracted at 2 dpi and subjected to real-time PCR quantification. All data show means standard deviations for triplicate samples. Datafor one representative experiment out of three are shown.

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higher than that from iDCs, indicating that the lack of HIV-1production from the nucleofected mDC-LPS was probably notdue to a low efficiency of transfection.

To measure the infectivity of HIV-1 generated frompNLAD8-nucleofected DCs, GHOST/R5 cells were infectedwith the supernatants of the nucleofected DCs. GHOST/R5cells are human osteosarcoma cells that express CD4 andCCR5. These cells contain a GFP gene under the control ofthe HIV-2 LTR promoter, which is expressed during HIV-1infection via Tat transactivation acting as an indicator of in-fection (11). HIV-1 infection (GFP expression) in GHOST/R5cells was measured using flow cytometry. Background levels ofGFP-positive GHOST/R5 cells (0.61%) were observed when thecells were infected with the supernatants from nucleofectedmDC-LPS, whereas 2% to 9% GFP-positive GHOST/R5 cellswere detected when the cells were infected with the superna-tants from other types of DCs (Fig. 6B). Thus, transfection ofHIV-1 proviral DNA can generate infectious HIV-1 in iDC,mDC-TNF-�, and mDC-CD40L but not in mDC-LPS, sug-gesting that there is a strong block to HIV-1 gene expression inmDC-LPS.

To investigate whether restricted HIV-1 gene expression in

mDC-LPS was specific for the HIV-1 LTR promoter, a GFPreporter driven by a cytomegalovirus (CMV) promoter wasused to examine exogenous gene expression in DCs. Varioustypes of DCs were nucleofected with pmaxGFP, a GFP expres-sion plasmid with a CMV promoter (a control for nucleofec-tion). GFP expression was measured by flow cytometry at 24 hposttransfection. Only 6% GFP-positive mDC-LPS were de-tected, compared with 47%, 34%, and 37% GFP-positiveiDCs, mDC-TNF-�, and mDC-CD40L, respectively (Fig. 6C).When relative GFP expression levels were compared, mDC-LPS showed fourfold less GFP expression than did other typesof DCs (Fig. 6D). Consistent results were obtained from fourindependent experiments using DCs generated from differentdonors (not shown). Together, these results suggest that im-paired gene expression in mDC-LPS contributes to the posten-try restriction of HIV-1 replication.

DCs efficiently transmit HIV-1 to CD4� T cells. To compareHIV-1 trans-infections of CD4� T cells mediated by varioustypes of DCs, viral transmission efficiency was examined usingsingle-cycle and replication-competent HIV-1. After incuba-tion with small amounts of HIV-1 for 2 h, DCs were washedand cocultured with a CD4� T-cell line (Hut/CCR5 cells) for2 days. Infection was determined by measuring the luciferaseactivities in cell lysates or Gag p24 levels in the supernatants at2 dpi. Samples from cultures of DCs alone were used as con-trols for HIV-1 cis-infection.

Compared with controls with DC alone, trans-infection ofHIV-1 pseudotyped with an R5 HIV-1 envelope (HIV-Luc/JRFL) in cocultures was enhanced 86-fold (P � 0.0001) bymDC-LPS, while iDCs, mDC-TNF-�, and mDC-CD40L me-diated 15-, 26-, and 3-fold (P � 0.05) increases in viral trans-mission, respectively (Fig. 7A). HIV-Luc/JRFL infection inmDC-LPS was strongly blocked (Fig. 7A and data not shown);however, upon coculture with CD4� T cells, mDC-LPS werethree- to fivefold more efficient than other types of DCs in theability to transmit HIV-Luc/JRFL to CD4� T cells (Fig. 7A).Consistently, low levels of HIV-1 cis-infection were detected inmDC-CD40L without coculturing them with CD4� T cells,which might help to explain the lower level of trans-infectionmediated by mDC-CD40L (Fig. 7A).

Similar results were obtained using replication-competentHIV-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-�, andmDC-CD40L, respectively (Fig. 7B). Relative to iDCs, varioustypes of mDCs appeared to transfer HIV-1 to CD4� T cells moreefficiently, despite the fact that there was no detectable viralreplication in mDC-LPS and mDC-TNF-� at 2 dpi (Fig. 7B). Lowlevels of HIV-1NLAD8 cis-infection were detected in iDCs andmDC-CD40L (Fig. 7B), indicating that DC-mediated trans-infec-tion is more efficient than cis-infection. Together, these resultssuggest that DCs potently mediate HIV-1 trans-infection, regard-less of their ability to support viral replication.

DISCUSSION

Elucidating the mechanisms underlying DC-mediated HIVinfection and viral transmission will enhance our understand-ing of HIV interactions with host cells (52). Here we provideevidence that cis- and trans-infections of HIV-1 mediated by

FIG. 5. Endocytosed replication-competent HIV-1 in DCs doesnot produce significant amounts of late RT products. (A) HIV-1 fusionis required for generating late RT products in DCs. DCs were infectedwith HIV-1NLAD8, washed, trypsinized, and cultured for 12 h beforethe cells were lysed for real-time PCR detection. (B) Enhanced HIV-1endocytosis and retention in mDC-LPS. DCs were infected with HIV-1NLAD8, washed, trypsinized, and cultured for 12 h before the cellswere lysed for Gag p24 detection. T-20 was present during the viralincubation and the 12-h culture. All data show means standarddeviations for duplicate or triplicate samples. Data for one represen-tative experiment out of three are shown.

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DCs are dissociable pathways. HIV-1 cis-infection is restrictedin mDC-LPS and impaired in mDC-TNF-�; however, thesecells efficiently promoted HIV-1 trans-infection of CD4� Tcells, suggesting that, in vivo, the various DC subsets facilitateHIV-1 dissemination to different extents via cis- and trans-infections. It has been proposed that DCs transfer HIV-1 toCD4� 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 afterde novo HIV-1 production by cis-infected DCs (46). However,our findings of dissociable cis- and trans-infections mediated byDCs suggest that these two processes are not necessarily se-quential or interdependent.

Our results highlight the potential significance of DC-medi-ated HIV-1 transmission in vivo. In submucosal tissues, HIV-

infected iDCs may act as a viral reservoir to further the spreadof infection. HIV-1 coinfection with other sexually transmittedpathogens may induce DC maturation through inflammatoryresponses, such as upregulated cytokine production (43).Given their proximity to CD4� T cells in lymphoid tissues,mDCs may potently stimulate HIV-1 transmission to theseactivated CD4� T cells. A recent study indicated that signifi-cantly increased plasma LPS levels in HIV-infected humanscorrelate with systemic immune activation and AIDS progres-sion (6). It is conceivable that increased LPS in HIV-infectedindividuals may induce DC maturation, thereby stimulatingHIV-1 dissemination in vivo. Similarly, more recent reportsindicated that LPS-activated CD34� cell-derived Langerhanscells mediate efficient trans-infection of HIV-1 (10, 14).Despite the convenience of using monocyte-derived DCs,

FIG. 6. Impaired gene expression in mDC-LPS contributes to the restriction of HIV-1 replication. (A) Gag p24 levels in DCs after nucleo-fection with HIV-1 proviral DNA. DCs were nucleofected with plasmid pNLAD8. Gag p24 in supernatants and cell pellets was measured at 5 dayspostnucleofection. (B) Nucleofection of mDC-LPS with HIV-1 proviral DNA failed to produce infectious HIV-1. GHOST/CCR5 cells wereinfected with the supernatants of DCs that had been nucleofected with pNLAD8 (at day 5 postnucleofection from the experiment described forpanel A). HIV-1 replication was quantified by measuring GFP expression at 3 dpi by flow cytometry. Culture medium was used as a mock-infectedcontrol. (C and D) Impaired gene expression in mDC-LPS. DCs were nucleofected with plasmid pmaxGFP (expression is controlled by a CMVpromoter). GFP expression was measured by flow cytometry at 24 h postnucleofection. (D) Mean fluorescence intensities of GFP from 1 � 104

DCs, measured by flow cytometry at 24 h postnucleofection. All data show means standard deviations for duplicate or triplicate samples. Datafor one representative experiment out of four are shown.

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these cells may not fully mimic the physiological functions ofthe DC subsets that are involved in HIV infection in vivo.Thus, further studies are required to confirm these in vitroobservations, using myeloid DCs, plasmacytoid DCs, Langer-hans cells, and autologous CD4� T cells from HIV-infectedindividuals.

DCs in vivo would likely be exposed to HIV-1 before orsimultaneously to a maturation stimulus. It has been reportedthat HIV-infected blood myeloid DCs and monocyte-derivedDCs fail to mature in culture (24, 25). Moreover, DCs fromindividuals with acute HIV infection have reduced expressionof the costimulatory molecules CD80 and CD86, and thismight influence DC-induced T-cell responses (31). These re-sults suggest that productive HIV infection of DCs underminesthe direct induction of T-cell-mediated immunity. In contrast,other studies have indicated that monocyte-derived DCs fromHIV-infected individuals can efficiently induce cytotoxic T cellsto respond to various antigens (15, 41). In addition, no func-

tional defects in cytokine production were observed followingstimulation of HIV-infected myeloid DCs and plasmacytoidDCs (45). In our study, to better compare HIV-1 infectionsamong various types of DCs, viral infection was performedafter generating mDCs from iDCs with different stimuli.

We found that distinct mechanisms contributed to postentryblocks of HIV-VSV-G and replication-competent HIV-1 inmDC-LPS. Thus, it is critical to consider the potential differ-ences in using HIV-VSV-G instead of authentic HIV-1 tostudy postentry infection. Although endocytosis of both typesof viruses was increased in mDC-LPS compared with that inother types of DCs, different viral trafficking pathways mightaffect the various steps in the viral life cycle. HIV-1-VSV-Genters cells through a low-pH-dependent endocytic pathway(1). Relative to those in other types of DCs, increased RT andefficient integration were detected in HIV-Luc/VSV-G-in-fected mDC-LPS, indicating that endocytosed HIV-VSV-Gcan complete uncoating, RT, and integration in mDC-LPS,while impaired gene expression in mDC-LPS could eventuallyblock viral expression of the luciferase reporter in the infectedcells. In contrast, in HIV-1NLAD8-infected mDC-LPS, re-stricted RT appeared to be an important component of thepostentry block, and impaired gene expression in these cellsmight also play a role. For LPS-induced mDCs, it has beenshown that HIV-1 is internalized into nonconventional, nonly-sosomal, acidic compartments (20), which may restrict the RTprocess of endocytosed HIV-1NLAD8.

In addition to the restriction of RT, it has been proposedthat there is a postentry block of HIV-1 in mDCs at the tran-scriptional level (4, 21). Using nucleofection of HIV-1 proviralDNA in DCs to overcome any restrictions in the early steps ofthe HIV-1 life cycle, we found that impaired gene expression inmDC-LPS contributed to the potent block of HIV-1 replica-tion in these cells. Moreover, we observed that LPS-treatedCD4� T cells showed 1.2- to 1.4-fold increases in the efficiencyof a single-cycle HIV-1 infection (not shown), suggesting thatLPS does not generally induce restriction of HIV-1 infection inpermissive cells. Consistent with this observation, an earlystudy demonstrated that LPS is a potent stimulator of HIV-1expression in monocytes and macrophages (39). The impairedHIV-1 gene expression in mDC-LPS might be due to ineffi-cient transcription and/or translation. A comprehensive geneexpression analysis showed that 225 transcripts among a totalof 31,837 tag sequences were statistically different betweeniDCs and mDC-LPS (26). Moreover, treatment of iDCs withLPS activates several signal transduction pathways, includingthe NF-�B pathway (2). Therefore, the impaired gene expres-sion in mDC-LPS is unlikely to be specific for HIV-1.

Although 2-LTR circles have been used as a marker fornuclear import of HIV-1 DNA, it is not always technicallycorrect to interpret the lack of 2-LTR circles as an indicator ofblocked nuclear import of viral DNA (57). Our data showedthat 2-LTR circles were undetectable in HIV-VSV-G-infectedmDC-LPS, while 1-LTR circles and integrated proviral DNAwere detected in these cells. This might be due to the lowabundance and degradation of 2-LTR circles in mDC-LPS. Ithas been reported that in HeLa-CD4 cells infected with HIV-1at 2 dpi, most viral DNA is fully processed into integratedprovirus (�55%) and 1-LTR circles (�35%), while only traceamounts of 2-LTR circles (�5%) can be detected (58). We

FIG. 7. DCs efficiently transmit HIV-1 to CD4� T cells. (A) DC-mediated transmission of single-cycle HIV-1. DCs (1 � 105) werepulsed with HIV-Luc/JRFL (MOI, 0.3) for 2 h, washed, and coculturedwith CD4� Hut/CCR5 T cells (1 � 105) for 48 h. Infection was deter-mined by measuring luciferase activities of the cell lysates. cps, countsper second. (B) DC-mediated transmission of replication-competentHIV-1. DCs (8 � 104) were pulsed with HIV-1NLAD8 (4 ng of p24) for2 h, washed, and cocultured with Hut/CCR5 cells (8 � 104) for 48 h.Infection was determined by measuring Gag p24 levels in supernatants.Samples with DC alone were used as controls for HIV-1 cis-infection.All data show means standard deviations for duplicate or triplicatesamples. Data for one representative experiment out of three areshown.

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estimated the abundance of LTR circles and integrants invarious types of DCs infected with HIV-VSV-G. Interestingly,only 0.1 to 1% of the late RT products were converted into2-LTR circles, while 0.3 to 22% and 4 to 35% of the late RTproducts were converted into 1-LTR circles and integratedproviral DNAs, respectively. These data suggest that the ratioof HIV-1 DNA to integrants might be cell type dependent.

The inefficient block of HIV-1 uptake into DCs by the fusioninhibitor T-20 suggests that HIV-1 entry into DCs occurs pri-marily through endocytosis rather than fusion. Nobile et al.reported that DC-SIGN, a C-type lectin molecule highly ex-pressed on monocyte-derived DCs, facilitates HIV-1 captureand intracellular accumulation but inhibits viral fusion (36).This may help to explain the low levels of HIV-1 fusion andreplication in DCs. We recently reported that mDC-LPS aremore efficient than iDCs at transmitting HIV-1 to various typesof target cells, independent of C-type lectins, including DC-SIGN (48). In addition, we observed that HIV-1 uptake bymDC-LPS was slightly increased (around 30%) by T-20 treat-ment for 2 to 12 h (Fig. 4A and 5B), suggesting that there is abalance between HIV-1 fusion and endocytosis. The blockadeof fusion-mediated HIV-1 entry by T-20 might decrease thedegradation of Gag p24 in the cytosol, thereby indirectly in-creasing the amount of internalized HIV-1 in vesicles of mDC-LPS. A similar compensatory link between HIV-1 fusion andendocytosis has been reported for CD4� primary T cells (42).In that study, blockade of CXCR4-tropic HIV virion fusionwith AMD3100, a CXCR4-specific entry inhibitor, increasedvirion entry into CD4� T cells via the endocytic pathway.

Although endocytosis-mediated HIV-1 entry can lead toproductive viral infection in certain cell types, including humanprimary macrophages (13, 32), our results suggest that efficientRT and productive HIV-1 infection in DCs require fusion-mediated viral entry. A recent report indicated that HIV-1enters Langerhans cells primarily by multiple-receptor-medi-ated endocytosis and that virions persist intact within the cy-toplasm for several days without productive replication (27).The majority of endocytosed HIV-1 confined in intracellularcompartments in DCs will eventually be degraded. In fact,rapid intracellular HIV-1 degradation has been reported forboth iDCs and mDCs (35, 37, 46, 48).

CD40L-induced mDCs appeared to be more susceptible toHIV-VSV-G infection than were other types of DCs. This waslikely due to more efficient nuclear import and integration ofviral DNA in mDC-CD40L, as more 1-LTR and 2-LTR circlesand proviruses were detected in the infected cells. In contrast,HIV-1NLAD8-infected mDC-CD40L and iDCs showed similarlevels 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 effi-cient in mDC-CD40L. It has been shown that productiveHIV-1 infection of DCs is triggered by CD40L treatment (16,23); however, other studies have reported that HIV-1 infectionis decreased in CD40L-induced mDCs (4, 34). These discrep-ant results might be due to different DCs and recombinantCD40L used in these studies.

Cellular restriction of HIV-1 infection in DCs may reflectinnate antiviral immunity of the antigen-presenting cells. It hasbeen reported that the antiretroviral factor APOBEC3G/3F(apolipoprotein B mRNA editing enzyme, catalytic polypep-

tide-like 3G and 3F) mediates postentry resistance to HIV-1infection in monocyte-derived iDCs (38). In addition, LPS-induced mDCs have reduced susceptibility to HIV-1 infection,which correlates with increased APOBEC3G levels (38). Arecent report suggests that langerin-mediated HIV-1 internal-ization results in viral degradation, thus inhibiting HIV-1 rep-lication in Langerhans cells (12). A better understanding ofintrinsic antiretroviral immunity in DCs will provide new in-sights into more effective intervention strategies against HIV-1infection and dissemination mediated by DCs.

ACKNOWLEDGMENTS

We thank Eric Freed and Stephen Hughes for critical readings ofthe manuscript. We thank Michael Emerman, Eric Freed, StephenHughes, and Vineet KewalRamani for the kind gift of reagents. Thefusion inhibitor T-20 was obtained from Roche through the AIDSResearch 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.

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