In a previous study of Vietnamese IDU EUs we identified some individuals whose CD4 T lymphocytes showed reduced susceptibility to in vitro infection by replicative strains of HIV-1 11. HIV-1 replication in CD4 T cells from three EUs (W276, W278 and B195) was far less efficient than in CD4 T cells from healthy controls. Analysis of the CCR5 gene in the same population revealed heterozygous mutations in some subjects 2425. In two cases (B184 and W336) the mutations in CCR5 were associated with reduced co-receptor function in transfected cell lines 2426, but the effect of these mutations in heterozygous primary cells was not assessed.

CD4 T cells from the five EUs studied here (W276, W278, B195, B184 and W336; Table 1) had reduced susceptibility to infectious strains of HIV-1 (data not shown and 11; see Materials and Methods for corresponding subjects designations). We used single-round infection with envelope-pseudotyped HIV-1 NL4.3Δ env particles bearing the luciferase reporter gene to investigate whether the reduced HIV-1 susceptibility of the EUs' CD4 T cells was detectable during the first cycle of viral replication (fig. 1). In four out of five cases (W278, B195, B184, W336), CD4 T cells were less susceptible to infection by a CCR5-tropic (R5) HIV-1 pseudotype (HIV-BaL), while they were permissive to infection by a CXCR4-tropic (X4) HIV-1 pseudotype (HIV-HxB2) and to HIV-1 particles pseudotyped with the G protein from vesicular stomatitis virus (HIV-VSVG), which has a ubiquitous (pantropic) receptor and uses an endocytic entry pathway 27 (fig. 1). These results showed that the CD4 T cells of the EUs with heterozygous mutations in CCR5 (B184 and W336) were less susceptible to infection by HIV-1 R5 pseudotype and confirmed our previous observation that HIV-1 restriction in subjects W278 and B195 is specific to R5 viruses 11. As shown, single-round infection of CD4 T cell from these four EUs was partially reduced. Interestingly, when replication-competent HIV-1 strains were used, inhibition of infection was much stronger (data not shown and 11), suggesting that the restrictions observed during the first round of HIV-1 replication are amplified during subsequent cycles.

Remarkably, CD4 T cells from subject W276 were resistant to both R5 and X4 HIV-1 pseudotypes and also to the HIV-VSVG pseudotype. These results indicated that the restriction was independent not only of HIV coreceptors, as previously shown 11, but also of the entry pathway used by the virus (fig. 1).

As already mentioned, CCR5 heterozygous mutations had been detected in subjects B184 and W336 (G106R and C178R respectively) 2425. These (or equivalent) mutations, when present in the homozygous state in transfected cell lines, affect the receptor conformation and both CCR5 membrane trafficking and function 2426. However, CCR5 surface expression by these two EUs' primary CD4 T cells has not been evaluated before.

Flow cytometry of R5-restricted CD4 T cells revealed that the percentage of CD4 T cells expressing detectable surface CCR5 was far lower in the two EUs carrying heterozygous CCR5 mutations (B184 and W336) than in controls expressing the wild-type (wt) CCR5 molecule (fig. 2A). In contrast, no such difference was found, in either the percentage (fig. 2A) or the mean fluorescence intensity (MFI), in the other two EUs (W278 and B195) who both had wt CCR5 (the CCR5 MFI was 1.38 and 1.21 in subjects W278 and B195, respectively, and 1.39 ± 0.24, mean ± SD, in five CCR5-wt controls).

Therefore, the low surface expression of CCR5 on CD4 T cells from EUs B184 and W336 is likely linked to CCR5 mutations and appears to affect R5 virus entry into target cells. However, HIV R5 replication in CD4 T cells from subjects W278 and B195 was restricted despite normal CCR5 surface expression (fig. 2A). We therefore examined whether CCR5 function was impaired in the CD4 T cells of these two subjects, affecting signaling events potentially involved in HIV-1 replication 2829.

As actin cytoskeleton reorganization is a major characteristic of chemokine responses, we analyzed CCR5-mediated actin polymerization in CD4 T cells from subjects W278 and B195. RANTES stimulation of CD4 T cells induced a rapid increase in the F-actin content of cells from the two EUs and from four CCR5-wt controls (fig. 2B). The peak responses occurred 15–30 s after stimulation, in keeping with a fully functional chemoreceptor 30. Cell pretreatment with the CCR5 inhibitor TAK-779 31 abrogated actin polymerization.

The restriction in CD4 T cells from subjects W278 and B195 affected HIV-1 viruses pseudotyped with different R5 tropic envelopes (JRFL 32 and YU2 33) (fig. 2C). These results confirmed that the restriction in W278 and B195 CD4 T cells is specific for the CCR5 entry pathway and indicated that it is independent of CCR5 expression and function.

R5 virus entry into CD4 T cells can be blocked by endogenously produced β-chemokines 20. We therefore investigated whether the restriction of HIV-1 R5 replication in CD4 T cells from subjects W278 and B195 could be overcome by neutralizing monoclonal antibodies (MAbs) to RANTES, MIP1α and MIP1β. The addition of anti-β-chemokine mAbs, but not of irrelevant IgGs, strongly enhanced the infection of both W278 and B195 cells by the HIV-1 pseudotype, while no significant enhancement was observed in CCR5-wt control cells with similar CCR5 surface expression (fig. 3A). These results suggest that endogenously produced β-chemokines may be responsible for the inhibition of HIV-1 infection in CD4 T cells from subjects W278 and B195.

When we challenged CD4 T cells with BaL pseudotyped HIV in a setting of weak β-chemokine production (before PHA and IL2 activation; <3 ng) we found that infection was as efficient in the two EUs as in controls (fig. 3B). This was consistent with the hypothesis that the inhibition of HIV-1 infection in PHA-activated CD4 T cells from subjects W278 and B195 involved chemokines produced upon cell activation. However, quantification of β-chemokines produced by PHA-activated CD4 T cells at the time of HIV-1 infection (three days after PHA stimulation) showed no significant increase in β-chemokine secretion in W278 and B195 cell cultures compared to controls (Table 2).

To investigate the possibility of enhanced sensitivity to β-chemokines, we infected non-stimulated CD4 T cells from subject B195 (not enough cells from subject W278 were available) in the presence of a cocktail of recombinant RANTES, MIP1α and MIP1β added at increasing concentrations. In the presence of low levels (≤ 5 ng) of recombinant β-chemokines, HIV-1 replication was comparable in B195 and CCR5-wt control CD4 T cells (Fig. 3C), as already observed in the absence of added β-chemokines (Fig. 3B). In contrast, when the β-chemokine levels were increased, the efficiency of infection fell sharply in B195 CD4 T cells and far less markedly in control CD4 T cells (fig. 3C) (ID₅₀ values were 8.12 ± 1.58 ng/ml and 59.34 ± 16.87 ng/ml for B195 and control respectively). CD4 T cell CCR5 surface expression was similar in the two individuals (data not shown). These results indicated that HIV infection of CD4 T cells from EU subject B195 was unusually susceptible to inhibition by β-chemokines.

The blockade of in vitro HIV infection in CD4 T cells from subject W276 was independent of viral tropism and of the entry pathway (fusion or endocytosis) (fig. 1). In a fluorimetric fusion assay with cells expressing HIV-1 envelope proteins 34, W276 CD4 T cells showed normal membrane fusion (not shown), further supporting post-entry restriction of viral replication in these cells.

Previous qualitative PCR analysis of viral replication in CD4 T cells from subject W276 suggested that the restriction step occurred before integration 11, but early times post-infection were not analyzed. We therefore used single-round infection and real-time PCR to determine the precise stage at which the restriction occurred. Early reverse transcription products (R-U5) were far lower in W276 cells than in control cells, from the very first hours after infection (fig. 4A), and they increased very little during the time course of infection. Levels of PCR products corresponding to subsequent replication steps were also decreased (not shown). These results suggest that early post-entry steps of viral replication, most likely involving reverse transcription, are impaired in W276 CD4 T cells. Note that W276 CD4 T cells were readily activated by PHA (>95% of cells were CD25+ at the time of challenge) thus discarding that the restriction of HIV-1 infection was caused by a defect of the response to PHA-stimulation. In addition, HIV-1 restriction in W276 CD4 T cells was not overcome by increasing the size of the inoculum of VSV-G pseudotyped HIV-1 (fig. 4B), arguing against a role of a saturable restriction factor.

To determine whether the restriction of viral replication in CD4 T cells from subject W276 was specific to HIV-1 or also affected other lentiviruses, we challenged the cells with SIVagm and SIVmac luciferase reporter viruses (fig. 4C) pseudotyped with VSV-G. Replication of both viruses was strongly inhibited in W276 CD4 T cells.

To determine whether resistance to infection was transient or persistent, we tested primary CD4 T cells or PBMC obtained from the five EUs at various times during 2–6 years of follow-up. The samples included cells obtained from four of the subjects (W276, W278, W336 and B184) after alleged interruption of IV drug use (Table 3). In infectivity assays with PHA-activated cells, HIV-1 replication was always inhibited in the EU cells compared to control cells. Moreover, the same pattern of HIV-1 inhibition (R5-restricted or tropism-independent) was observed in serial samples from each EU (not shown), confirming the persistence of individual restriction phenotypes.

The five EUs studied here (Table 1) belonged to a population of intravenous drug users (IDU) who had been exposed to HIV-1 through needle sharing for many years 1153. Subjects W276, W278, and B195 correspond to subjects EU1 to EU3 and subject B184 corresponds to subject EU13 in 11. W336 was first described in 25. When recruited, they had been using drugs for 17 to 26 years. All continued high-risk practices for several years despite medical counseling. Four subsequently said they had stopped at-risk drug use between 2000 and 2003 (Table 1). Subject B195 was lost to follow-up in July 1999. Controls were Vietnamese (20) and European (7) healthy blood donors with a low risk of HIV-1 infection (Red Cross, Vietnam and Centre de Transfusion Sanguine Ile-de-France, Rungis, France). All the infectivity assays with EU CD4 T cells were performed in parallel with susceptible CD4 T cells from at least three randomly selected controls. All participants gave their informed consent.

Peripheral blood mononuclear cells (PBMC) from EUs and controls were isolated from whole blood by Ficoll-Hypaque centrifugation. CD4 cells were purified from thawed PBMC by positive selection with antibody-coated immunomagnetic beads (Miltenyi Biotech, France). Activated CD4 T cells (>95% CD4+CD3+CD25+ as estimated by flow cytometry) were obtained after stimulation for three days with phytohemagglutinin (PHA, 1 μg/ml) and interleukin-2 (IL2) (Chiron, France, 100 IU/ml) and were maintained in RPMI 1640 medium containing 10% fetal calf serum, penicillin/streptomycin (100 U/ml) and IL2.

Pseudotyped reporter retroviral particles were produced by transiently co-transfecting 293T cells with the proviral constructs pNL-Luc-E-R+, pSIVmac-Luc-E-R+ or pSIVagm-Luc-E-R+ 4354 and the VSV-G, HxB2-Env, BaL-Env, JRFL-Env or YU2-Env expression vectors (7.5 μg each) using the lipofection reagent SuperFect (Qiagen, France). Supernatants were harvested 48 h after transfection, and 10⁵ CD4 T cells were infected (m.o.i: 0.1–1.0) in triplicate in 96-well plates with a spinoculation protocol 55 (1 hour of centrifugation at room temperature at 1500 g, followed by 1 hour at 37°C). After challenge, cells were extensively washed and then cultured.

Three days after challenge the cells were harvested and lysed with 100 μl of luciferase lysis buffer (Promega, France). Luciferase activity was quantified in 10 μl of each lysate with the Promega Luciferase Assay System in a Veritas microplate luminometer (Turner BioSystems, CA, USA).

DNA was extracted from PBMC with the DNeasy Tissue Kit (Qiagen, Courtaboeuf, France). The full-length coding region (exon 4) of the CCR5 gene was amplified with primers and in conditions described elsewhere 24.

PCR products were purified with the ExoSAP-IT^® enzyme for PCR Product Clean-Up (Pharmacia-Amersham, USA) and were directly sequenced with the BigDye Terminator cycle sequencing kit (ver.3.1; Applera, France). Sequences were determined with an automatic sequencer (ABI-Prism 3100, Applied Biosystem, USA) and analyzed with SeqScape software version 2.5 (Applied Biosystem, USA).

Ten days after PHA activation, CD4 T cells were incubated for 30 minutes at room temperature with CCR5-FITC (clone 2D7) (BD Bioscience, France) and analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter, Paris, France).

Actin polymerization in CD4 T cells was measured as described elsewhere 30. Briefly, ten days after PHA stimulation, cells (1 × 10⁷ cells/mL) were incubated in RPMI medium containing 20 mM HEPES in the presence or absence of inhibitor. RANTES (30 nM) was then added to the cell suspension. At each indicated time point (15 s to 2 min), a 50-μL aliquot of cell suspension was mixed with 200 μL of labeling buffer consisting of 10^-7 M FITC-phalloidin (Sigma), 0.125 mg/mL L-α-lysophosphatidylcholine palmitoyl (Sigma) and 4.5% PFA in PBS. The kinetics of actin polymerization was monitored by means of flow cytometry. Results are expressed as follows: [MFI after addition of ligand/MFI before addition of ligand] × 100]. MFI values before ligand addition were arbitrarily set at 100%. Owing to the large number of cells required, CD4 T cells were amplified on irradiated heterologous feeder PBMC for two weeks prior to testing. The pattern of HIV-1 restriction in amplified cells was similar to that found in the primary CD4 T cells (not shown). TAK-779 was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

Levels of β-chemokines, RANTES, MIP-1α and MIP-1β in the supernatants of CD4 T cells were measured after 72 h of culture with or without PHA stimulation, by using commercial ELISA kits (Quantikine, R&D systems, France).

Three days after PHA stimulation, CD4 T cells were challenged with DNase (Invitrogen, France)-pretreated viruses (1 h at room temperature). At the times indicated, 5 × 10⁵ cells were washed in PBS and lysed, then total DNA was extracted with the DNeasy Tissue Kit (Qiagen, France). Early HIV-1 reverse transcription products were quantified with an ABI PRISM 7000 instrument (Applied Biosystems, France) using specific primers and probe as previously described 56. One hundred nanograms of template DNA was used per reaction, and the albumin gene was used as a housekeeping gene to normalize sample input. 8E5 cells containing one integrated copy of HIV-1 per cell 57 were used to construct standard curves.