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CORRESPONDENCE Vincent Piguet: vincent.piguet{at}medecine.unige.ch
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, enhanced HIV-1 infection of iDCs, indicating that A3G/3F controls the sensitivity of iDCs to HIV-1 infection. Furthermore, sequences of HIV reverse transcripts revealed G-to-A hypermutation of HIV genomes during iDC infection, demonstrating A3G/3F cytidine deaminase activity in iDCs. When we separated the fraction of iDCs that was susceptible to HIV, we found the cells to be deficient in A3G messenger RNA and protein. We also noted that during DC maturation, which further reduces susceptibility to infection, A3G levels increased. These findings highlight a role for A3G/3F in explaining the resistance of most DCs to HIV-1 infection, as well as the susceptibility of a fraction of iDCs. An increase in the A3G/3F-mediated intrinsic resistance of iDCs could result in a block of HIV infection at its mucosal point of entry.
, Trim5; VSVG, vesicular stomatitis virus G.
The spread of HIV-1 over time has occurred mainly through sexual transmission. This transmission necessitates that minute amounts of virus traverse mucosal surfaces to reach replication-competent sites in lymphoid tissue. Model systems have indicated that one of the key events during this transmission is the transfer of HIV infection from DCs to CD4+ T cells (for review see references 1–3).
HIV infects DCs in vitro only when relatively high viral inocula are used (for review see reference 1) (4–6). Presently, the mechanism responsible for the poor replication of HIV in immature DCs (iDCs), the type of DCs that likely encounters HIV in the mucosal tissues during sexual transmission, has not been elucidated.
In the absence of viral replication, DCs also can capture and transfer viral material to CD4+ T cells via an infectious synapse, which results in vigorous infection (7–10). The contribution of this "capture–transfer" pathway remains unclear in vivo.
Langerhans cells/DCs replicate at low levels, preferentially HIV-1 R5 compared with HIV-1 X4 (5, 11, 12). This preferential replication of R5 strains versus X4 strains appears to be caused in part by levels of coreceptors on the surface of iDCs and is also partly due to a block during viral fusion with iDCs (11, 12). However, infection of iDCs is still inefficient, irrespective of the viral env, when compared with activated CD4+ T cells (for review see reference 1).
In this study, we asked whether iDCs expressed an env-independent intrinsic resistance to HIV-1 infection that could operate after viral entry. The idea of intrinsic resistance derives from the discovery of cellular restriction factors to HIV-1 infection (for review see reference 13). Two families of restriction factors in human cells have been recently identified: the TRIM family and the APOBEC family (14, 15). Specifically, one member of each family can operate, at least in some circumstances, after viral entry and before viral integration into the nucleus (e.g., TRIM5 [T5] and APOBEC3G [A3G]) (15, 16). Human T5 restricts HIV-1 only weakly (15), whereas A3G effectively restricts HIV-1 after entry in primary cells such as resting CD4+ T cells (16). In fact, A3G is a potent host antiretroviral factor that can restrict HIV-1 infection through at least two mechanisms. First, A3G is a DNA deaminase that is incorporated into virions during viral production and subsequently triggers massive G-to-A hypermutation in the nascent retroviral DNA (17, 18). This mode of action is counteracted by Vif that prevents incorporation of A3G into virions (19). Second, cellular A3G can function as a post-entry restriction factor for HIV during reverse transcription in resting CD4+ T cells (16), where it resides in a low molecular mass (LMM) active form. This second mechanism does not seem to be counteracted by Vif, because incoming viral particles contain little Vif. The potential contribution of these cellular restriction factors to the low levels of HIV-1 infection in iDCs has not been tested. In this paper, we demonstrate an env-independent restriction to HIV-1 infection in human iDCs using single-round infection assays with viral particles pseudotyped with vesicular stomatitis virus G (VSVG). We will demonstrate a role for A3G, and to a lesser extent for APOBEC3F (A3F), in restriction of HIV-1 infection in DCs.
RESULTS
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ABSTRACT
RESULTS
DISCUSSION
MATERIALS AND METHODS
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HIV-1 replication is restricted in iDCs
We generated iDCs after 6 d of culture of monocytes in GM-CSF and IL-4, as previously described, and verified that the iDCs expressed the expected markers (i.e., CD14– and MHC class II+) (9, 20). To overcome the entry block that has been described in iDCs, we used single-round infection assays with HIV-1 
env and pseudotyped with VSVG (HIV-VSVG; Fig. 1 A).
Similar results were obtained using a lentiviral vector (LV) that is deleted in env, vif, vpr, vpu, and nef (LV-VSVG), indicating that the low susceptibility of iDCs to HIV infection was independent of these virulence genes (Fig. 1 B). iDCs were at least 50–100 times less susceptible to HIV-VSVG infection than Jurkat CD4+ T cells (JTs; Fig. 1, A–C). HIV-VSVG infection was equally efficient in activated peripheral blood lymphocytes and in JTs (unpublished data).
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, are active restriction factors in iDCs. Monocyte-derived DCs (mDCs) were transfected with small interfering RNA (siRNA) sequences specific for the target gene using lipofectamine reagent. The extent of down-regulation was monitored by Western blotting followed by densitometry analysis. Double transfection of a mix of three siRNA targeting A3G induced an efficient knockdown in 293T cells stably expressing A3G-HA (293T-A3G-HA cells; 80% down-regulation; Fig. 2 A) or in iDC (60–80% down-regulation; Fig. 2 B), which is an efficiency comparable to that reported for RNA interference of A3G in resting CD4 T cells (16).
In 293T-A3G-HA cells, siRNA-mediated interference of A3G did not increase HIV-1 infection (Fig. 2 C), but HIV infection was enhanced >7-fold (range = 2–22-fold, depending on the donor) in A3G knockdowned iDCs in comparison with control cells treated with equal amounts of siRNA targeting an irrelevant sequence (Fig. 2 D). Based on these results, we conclude that HIV-1 infection in iDCs is restricted at least in part by A3G.
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To substantiate the involvement of A3G/3F proteins in the early block observed in DCs, we analyzed the sequences of viral reverse transcripts 8 h after infection (Fig. 2 E). A substantial fraction of the incoming viral DNA molecules (
17%) harbored G-to-A mutations, the hallmark of an A3G/3F action.
Because T5 is another factor that can restrict HIV-1 infection in several cell types (15, 23), we used RNA interference to knock down human T5 in HeLa stably overexpressing T5-HA (HeLa-T5-HA; 80% down-regulation of T5; Fig. 3, A and B, left) and in iDCs (80% down-regulation of T5; Fig. 3, A and B, right).
Although RNA interference of T5 in HeLa-T5-HA relieved the block imposed to murine leukemia virus–N in human cells as expected (Fig. 3, C and D, left), interference of endogenous human T5 in iDCs did not increase HIV-VSVG infection (Fig. 3, C and D, right). Thus, human T5 is not a potent cellular restriction factor to HIV infection in iDCs.
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on GFP+ and GFP– cells. Strikingly, A3G mRNA was severely decreased in GFP+ permissive iDCs in comparison with GFP– iDCs, whereas A3F and T5 mRNA were equally present in both fractions of iDCs (Fig. 4 B). Tubulin was used as a loading control, indicating that this result was not explained by differences in cell counts between the GFP+ and GFP– populations. We also performed a Western blot analysis. A3G protein levels were decreased in the GFP+ iDCs in comparison with GFP– iDCs (Fig. 4 C). Because HIV Vif does not affect A3G mRNA in infected cells (19), this result suggests that the susceptibility of a fraction of iDCs to HIV-1 is caused by the absence of A3G mRNA before infection of these cells.
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5–10-fold (Fig. 5 A).
We next quantified A3G expression by Western blot analysis in iDCs and mDCs. mDCs expressed three to fivefold more A3G than iDCs (Fig. 5 B). A3F antibodies did not function for Western blot analysis, thus precluding this type of analysis for A3F in mDCs (unpublished data). RNA interference of A3G in mDCs was inefficient, perhaps because of the low capacity of mDCs for internalization, which prevented us from directly testing the capacity of A3G to control HIV-1 infection in mDCs. Nevertheless, these results suggest that DC maturation further restricts HIV-1 infection through an increase in A3G expression.
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DISCUSSION |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We therefore considered the two families of restriction factors (TRIM/APOBEC) that have been identified in human cells (for review see reference 13). Among their family members, T5 and A3G are known to block HIV-1 after viral entry in select cells. T5 did not operate as a strong restriction factor in iDCs. This is not surprising given the fact that human T5 restricts HIV-1 only weakly in human cells (15). On the other hand, human A3G does function as a potent post-entry cellular restriction factor for HIV-1 in resting CD4+ T cells (16). A3G acts through cytidine deaminase activity (17, 18), as well as through less well-understood nonenzymatic mechanisms (26).
Three separate lines of evidence indicated that A3G could restrict HIV-1 infection in iDC. First, RNA interference of A3G in iDCs considerably increased HIV-1 infection in these cells. Interestingly A3F down-regulation also enhanced HIV infection in iDCs, though to a lesser extent than A3G down-regulation, suggesting that both A3G/3F may contribute to restricting HIV infection in iDCs. To confirm the involvement of A3G/3F in the early block of HIV infection in DCs, we analyzed the sequences of viral reverse transcripts. A substantial fraction of the incoming viral DNA molecules (17%) harbored G-to-A mutations, the hallmark of an active cytidine deaminase like A3G/3F, and these hypermutations harbored the signatures of both A3F and A3G action. Based on the known respective frequencies of their different signatures (21), it can be deduced that both enzymes contribute comparably to the total mutations found in reverse transcripts. It appears, however, that A3G is probably the main contributor to the restriction, because its RNA interference–mediated ablation leads to the strongest relief of the block in iDCs.
Second, iDC that were infectable with HIV-VSVG did not express A3G mRNA or A3G protein. Therefore, our result indicates that low A3G mRNA in a subset of iDCs possibly increases permissivity to HIV-1. Interestingly, A3F was expressed in both infected and uninfected iDC populations, which correlates with its less important contribution to the restriction of HIV infection in iDCs, as determined by RNA interference studies.
Third, we tried to modulate A3G expression and function in DCs by inducing DC maturation. Unlike resting CD4+ T cells that become permissive upon activation (16), DCs are more restrictive after maturation/activation. Interestingly A3G levels were up-regulated in mDCs, further indicating a link between A3G and HIV-1 infection in iDCs.
The recent description of A3G in an enzymatically active LMM form in resting CD4+ T cells and monocytes (16, 27) suggests that A3G possibly exists in that active LMM form in DCs as well. We recapitulated a simplified version of the assay in which we separated A3G SN that behaved similarly to A3G LMM. Our results demonstrated that the presence of A3G SN correlated with restriction. Monocytes and resting CD4+ T cells had the highest quantity of SN A3G and were not infectable. 293T and activated CD4+ T cells had low amounts of A3G SN and were completely permissive. Interestingly, mDCs had more A3G SN than iDCs. Collectively, our results indicate that A3G restriction explains, at least in part, the post-entry block observed in iDCs. Attempts to modulate A3G/A3F function in DCs could offer the potential to counteract HIV infection before its dissemination during the early events of HIV-1 transmission.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Preparation of primary mDCs.
Monocyte isolation from buffy coats of healthy donors (obtained according to guidelines of the ethical committees of the University of Geneva and the Rockefeller University) and subsequent generation of iDCs was performed as previously described (20). DC maturation was induced by 48 h of LPS stimulation at 20 ng/ml (Escherichia coli, strain 055:B5; Difco).
Cell culture reagents.
HeLa-T5-HA cells and 293T-A3G cells (a gift of D. Trono, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) were grown and maintained in DMEM with 10% FCS, 100 µg/ml penicillin and streptomycin, and 2 mM glutamine.
RNA interference in iDCs.
mDCs were transfected with siRNA sequences specific for the target gene using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations, with modifications available in Supplemental materials and methods.
Sequencing of viral DNA.
iDCs were infected with DNase-treated HIV-VSVG-GFP virions at a multiplicity of infection (MOI) of 10. Viral reverse transcripts were extracted with the DNeasy kit (QIAGEN) 8 h after infection and individually amplified by standard PCR using O.nef1 and O.U5.1 primers (see Supplemental materials and methods). They were then cloned with a cloning kit (TOPO-TA; Invitrogen) and sequenced. Finally, Clone Manager software (version 8; Scientific & Educational Software) was used for the alignments.
RT-PCR from iDC-infected cells.
iDCs were infected with HIV-VSVG expressing GFP (2 ng p24 gag/105 cells) for 5 d. Cells were sorted as GFP+ (infected) or GFP– (noninfected). Total RNA was extracted with TRIZOL (Invitrogen). RT-PCR was performed with 0.5 µg RNA using SuperScript one-step RT-PCR (Invitrogen). PCR for A3G and T5 was performed by 30 cycles of amplification at 94°C for 15 s, 55°C for 30 s, and 72°C for 1 min, or at 94°C for 15 s, 48°C for 30 s, and 72°C for 1 min, respectively, with gene-specific primers (see Supplemental materials and methods).
A3G subcellular localization.
Cells (see Supplemental materials and methods) were lysed with ice-cold lysis buffer (125 mM NaCl, 50 mM Hepes, pH 7.4, 0.2% NP40, 1 mM dithiothreitol, 0.1 mM PMSF, EDTA-free protease inhibitor cocktail; Sigma-Aldrich) for 30 min and centrifuged at 35,000 rpm (MC M150GX; Sorvall) for 1.5 h. SN and P were separated, and P was resuspended in a volume equal to that of SN and sonicated for 20 s (Sonic B12; Branson). Equal volumes of P and SN were loaded on gel and analyzed by standard Western blotting. Protein expression was quantified by densitometry analysis of specific bands. Ratios of A3G SN were calculated as a ratio of A3G SN/P, where the A3G P fraction had an arbitrary value of 1 based on densitometry analysis.
Online supplemental material.
Supplemental materials and methods contains information about virus stocks, RNA interference in iDCs, siRNA and primer sequences, antibodies, and A3G subcellular localization. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20061519/DC1.
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Acknowledgments |
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This work was supported by the Geneva Cancer League, the Leenaards Foundation, and the Swiss National Science Foundation (V. Piguet), and National Institutes of Health grant AI40045 (to R.M. Steinman). This work was also supported by the Human Science Frontier Program (V. Piguet and R.M. Steinman).
The authors have no conflicting financial interests.
Submitted: 18 July 2006
Accepted: 13 November 2006
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and 
, GFP expression in JTs; 
and 
, GFP expression in iDCs.
