Chemokine receptors CCR5 and CXCR4, together with CD4, are the major coreceptors for HIV-1 or HIV-2 infection [1–7]. For HIV-1, CCR5 usage corresponds to the slow/low, non-syncytium-inducing phenotype in previous classifications of primary HIV-1 isolates, whereas isolates with a rapid/high, syncytium-inducing phenotype use CXCR4 . HIV-2, like HIV-1, has been phenotypically divided into rapid/high or slow/low phenotypes . Several observations indicate that in about 50% of HIV-1-infected individuals CXCR4-tropic HIV-1 variants emerge during the course of infection preceding an accelerated CD4 cell decline and a more rapid disease progression [10–12]. A similar correlation between disease state and coreceptor use seems to exist for HIV-2 . However, the role of chemokine receptor usage in viral pathogenesis remains unclear. Recent studies indicate that an expanded coreceptor repertoire of HIV-1 is not a prerequisite for a progressive clinical course of HIV-1 infection [13,14]. CD4-independent infection has also been observed for many HIV-2 and SIV isolates [15–17] and has been suggested to influence viral tropism and pathogenesis.
To define the role of coreceptor usage in viral pathogenesis, a number of investigators have chosen non-human primates models of lentivirus infection. To date, HIV-2 is the only lentivirus of human origin that can reproducibly cause AIDS in non-human primates [18–20]. We previously described such an isolate, HIV-2/287, which induces rapid CD4 cell depletion and AIDS-related symptoms in 100% of infected Macaca nemestrina [19,21,22]. This virus was established upon serial passages of HIV-2 EHO, which was originally isolated in France from an AIDS patient from Ivory Coast . Through serial passages in vivo, HIV-2/287 acquired greatly enhanced pathogenicity in M. nemestrina as compared to the parental virus HIV-2 EHO [19,21]. These viruses therefore represent a useful model to study viral factors of HIV pathogenesis. In the present study, we investigated the replicative capacity and the role of CD4 and chemokine coreceptor usage in the pathogenicity of HIV-2/287 in M. nemestrina.
Materials and methods
Macaque and human peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood and depleted for CD8 cells using antibodies to CD8 (G10.1) and complement. The CD8-depleted PBMC were stimulated with phytohemagglutinin (PHA; 2 mg/μl) and cultured for 3–5 days in RPMI 1640 containing 10% fetal calf serum (FCS), penicillin (pen; 100 U/ml) and streptomycin (strep; 100 μg/ml), and interleukin-2 (IL-2; 20 U/ml). The human myelomonocytic U937 cell line was maintained in RPMI 1640 containing 10% FCS and pen/strep. The human osteosarcoma CD4-negative HOS cells transfected with CCR5 or CXCR4 were maintained in Dulbeccos's modified Eagle's medium (DMEM; Life Technologies Inc., Rockville, Maryland, USA) containing 10% FCS, antibiotics, and puromycin (1 μg/ml). The CD4-positive HOS cells were cultured in DMEM with 10% FCS, pen/strep, geneticin (500 μg/ml) and puromycin (1 μg/ml). The human osteosarcoma cell line GHOST and the GHOST-derived lines expressing CCR1, CCR2, CCR3, CCR4, CCR5, CXCR4, GPR15 and STRL33  were maintained in DMEM containing 10% FCS, pen/strep, geneticin (500 μg/ml) and hygromycin (100 μg/ml) for the parental GHOST, and the same medium supplemented with puromycin (1 μg/ml) for the cells expressing coreceptor; cells were split twice a week. For infection experiments, cells were seeded in 12-well plates 1 day prior to infection to obtain a subconfluent cell layer by the time of infection.
HIV-2 EHO stock was generated in the U973 cells and HIV-2/287 stock was generated in primary human lymphocytes. The U973 cells were infected with HIV-2 EHO in the presence of polybrene (2 μg/ml) and cultured in RPMI 1640 medium containing 10% FCS and pen/strep. Human PBMC, depleted for CD8 cells and stimulated with PHA (2 μg/ml) were cultured for 3–5 days in RPMI 1640 containing 10% FCS and 10% (20 U/ml) native IL-2 before infection with HIV-2/287. Viral stocks were collected at the peak of p27 antigen production. Cell-free virus was harvested from the cultures by low-speed centrifugation for 5 min to remove the majority of cells, followed by filtration through a 0.22 μm filter. Aliquots of virus were stored at −70°C.
p27 antigen assay
The p27 antigen capture enzyme-linked immunoabsorbent assay was performed according to manufacturer's instructions (RETRO-TEK, Zepto-Matrix, Buffalo, New York, USA).
GHOST4 cells were infected with different dilutions of virus supernatant in the presence of 20 μg/ml polybrene. After 1 h at 37°C, the virus was removed and the culture were maintained in 2 ml per well DMEM containing 10% FCS for 2 or 3 days before immunostaining. The experiments were performed during the first 2 weeks after the cells were thawed to avoid the decrease in the expression level of chemokine coreceptors that can occur at later passages.
At 2 or 3 days post-infection, medium was removed and the GHOST4 cells were washed twice in phosphate-buffered saline (PBS), before fixing with methanol. After removal of the methanol, the cells were washed once with PBS and incubated for 1 h at 37°C with PBS containing the primary antibody (serum from HIV-2/287-infected monkey; 1 : 200). The monolayer was then washed twice with PBS and incubated for 1 h at 37°C with the second antibody, peroxidase-conjugated mouse anti-human IgG (Sigma, St Louis, Missouri, USA; 1 : 1000). After two washes with PBS, the substrate, 3-amino-9-ethylcarbazole was added for 30 min at room temperature. Infected cells stained red within 15 min of the addition of substrate.
Inhibition of virus infection
One million macaque or human PBMC were incubated with: peptide TW70 (5 μg/ml; derived from T22, ); a cocktail of RANTES (500 ng/ml), macrophage inflammatory protein (MIP)-1a (500 ng/ml), and MIP-1b (500 ng/ml); an anti-CXCR4 monclonal antibody (MAb) 12G5 (20 μg/ml); or an isotype-matched IgG2a control antibody (20 μg/ml) for 30 min at 37°C before infection. Macaque PBMC isolated from three different animals were infected with a multiplicity of infection (m.o.i.) of 1 infectious unit per cell for HIV-2 EHO and an m.o.i. of 10−2 infectious units per cell for HIV-2/287. Human PBMC were infected with an m.o.i. of 10−2 infectious units per cell for HIV-2 EHO and HIV-2/287.
Detection of reverse transcriptase (RT) intermediates
CD8 cell-depleted, PHA-stimulated macaque and human PBMC were infected with 50 ng p27 equivalents of HIV-2 EHO or HIV-2/287 per 1 × 106 cells at 37°C for 1 h. Before infection, virus inocula were treated with RNase-free DNase I (Gibco) at concentration of 50 U/ml for 30 min at 37°C in the presence of 10 mM MgCl2 to remove any HIV-2-related DNA that might contaminate subsequent preparations. Heat-inactivated (60 min, 60°C) HIV-2 EHO and HIV-2/287 were included in each assay to control for the efficacy of this treatment. Some infections were performed in the presence of 10 μM zidovudine (ZDV; Sigma). The infected cells were washed with medium four times to remove residual free virus. Total DNA for amplification was prepared using a QIAGEN Kit (Qiagen, Valencia, California, USA). The DNA concentration of each sample was quantified by spectrophotometry. One microgram of DNA from each sample was amplified in a PCR mixture containing 0.2 μm each primer, 200 μM each of the four deoxynucleoside triphosphates, PCR buffer (PE/Applied Biosystems, Foster City, California, USA), 1.5 mM MgCl2, and 1.0 U Taq polymerase in a final volume of 50 μl. The reaction was subjected to one cycle of denaturation at 94°C for 7 min followed by 30 cycles of denaturation for 1 min at 94°C, annealing for 2 min at 60°C, and elongation for 4 min at 70°C.
The oligoprimers used for HIV-2 DNA detection were derived from the nucleotide sequence of HIV-2 EHO . The primer pair R (5′-GTAGAGCCT GGGTGTTCCCTGCTA-3′; sense) and U5 (5′-CCT AACAGACCAGGGTTTCTAGTG-3′; antisense) was used to amplify a 190 bp early RT product; the primer pair U5 (5′-CACTAGAAACCCTGGTCTGTTAGG-3′; sense) and GAG (5′-TGACAAGACGGAGCCTC TCGCGCC-3′; antisense) was used to amplify a 321 bp late RT product. A pair of primers complementary to the human β-globin gene, PCO4 54-73 and GH20 195-176 (PE/Applied Biosystems), was run separately in most experiments. One oligonucleotide of each complementary pair was 5′-end labeled with [32P]ATP by polynucleotide kinase (Gibco). The 32P-labeled PCR products obtained by amplification were analyzed by electrophoresis on 8% non-denaturing polyacrylamide gels, exposed to X-Omat AR (Eastman Kodak, Rochester, New York, USA) films with an intensifying screen at −70°C.
HIV-2 EHO and HIV-2/287 preferentially use CXCR4
We examined the coreceptor usage of HIV-2 EHO and HIV-2/287 by infecting GHOST4 cells expressing CCR1, CCR2, CCR3, CCR4, CCR5, CXCR4, GPR15 or STRL33. Fig. 1 shows that both HIV-2 EHO and HIV-2/287 use preferentially CXCR4. Although both viruses were able to infect GHOST4 cells expressing a number of coreceptors, HIV-2 EHO showed a broader coreceptor usage. The results in Fig. 1 also revealed that HIV-2/287 had a titer 10-fold lower than that of HIV-2 EHO in GHOST4 cells expressing CXCR4. As equivalent amounts of virus (determined by p27 antigen) were used in these studies, the lower levels of HIV-2/287 output [focus forming units (f.f.u.)/ml]; as compared to HIV-2 EHO indicated reduced infectivity for HIV-2/287 in these cells. Results obtained with other indicator cell lines, U373-MAGI expressing CXCR4 or CCR5, confirmed the CXCR4 tropism for both viruses and the lack of CCR5 usage for HIV-2/287 in these cells (data not shown).
CXCR4 is the predominant coreceptor used by HIV-2 EHO and HIV-2/287 to infect human and macaque cells
To determine the specific coreceptor used by HIV-2 EHO and HIV-2/287 to infect the human and macaque PBMC in vitro, we tested the effect of receptor-specific reagents on virus infection. These reagents included a peptide that binds to CXCR4 (TW70), a MAb against CXCR4 (12G5), and a cocktail of RANTES, MIP-1α and MIP-1, natural ligands of the β-chemokine receptors CCR1 to CCR5 (Fig. 2). The specificity of these reagents was confirmed by control experiments using HIV-1 LAI and HIV-1/162 (data not shown). The reactivity of MAb 12G5 with the macaque PBMC was verified by fluorescent cell antibody sorting and the results indicated an equivalent percentage of positive cells for macaque and human PBMC 3 days after stimulation with PHA (data not shown). The data in Fig. 2 show that HIV-2 EHO and HIV-2/287 infections were not inhibited by the mixture of RANTES, MIP-1α and MIP-1β, indicating that these viruses did not use CCR5 to infect human and macaque cells. The increase of infection observed for the two viruses in macaque PBMC in the presence of RANTES is reminiscent of what has been described for X4 tropic HIV-1 in human PBMC . However, the effect in human PBMC was observed with higher concentrations of RANTES, indicating that macaque PBMC may be more sensitive to this chemokine than human cells. In contrast to the results with β-chemokines, CXCR4-specific peptide TW70 and MAb 12G5 inhibited HIV-2 EHO and HIV-2/287 infection, indicating that these viruses use mainly the coreceptor CXCR4 to infect human and macaque cells. These results suggested that other coreceptor usage determined in transfected GHOST4 cells probably does not play a major role in the infection of human or macaque PBMC by these viruses.
HIV-2 EHO and HIV-2/287 are CD4-dependent viruses
To test dependence on CD4 for viral entry, we infected CD4-negative and CD4-positive HOS cells expressing CXCR4 or CCR5 with HIV-2 EHO and HIV-2/287. Neither virus was able to infect the CD4-negative cells, whereas the positive results obtained with CD4-positive HOS cells confirmed that both viruses are able to use CXCR4 and CCR5 (data not shown). These results demonstrated that both HIV-2 EHO and HIV-2/287 are CD4-dependent viruses.
Reduced infectivity and p27 production in HIV-2 EHO-infected macaque PBMC
The infectivity of each virus stock was determined by 50% tissue culture infectious dose (TCID50)  and by p27 production in macaque and human PBMC. The titer of HIV-2 EHO in macaque PBMC was 1.8 × 102 TCID50 (data not shown), significantly less than its titer of 5.9 × 107 TCID50 (data not shown) in human PBMC. In contrast, there was no significant difference in the titers of HIV-2/287 obtained in macaque or human PBMC (5.9 × 105 versus 1.8 × 105 TCID50, respectively). The higher titer of HIV-2/287 in macaque PBMC indicates that the adaptation of HIV-2 EHO in M. nemestrina resulted in a virus, HIV-2/287, capable of overcoming the growth restrictions in macaque PBMC.
The growth restriction for HIV-2 EHO in macaque PBMC was also demonstrated by p27 antigen production. Fig. 3 shows the kinetics of p27 production for HIV-2 EHO and HIV-2/287 infection in macaque and human PBMC. Infection of macaque PBMC with HIV-2 EHO resulted only in low levels of p27 viral antigen, with a plateau of 250 ng/ml occurring 3 days after infection with no subsequent increase. In contrast, HIV-2 EHO infection of human cells resulted in efficient core-antigen production at 1500 ng/ml with a peak at 3 days post-infection. These results indicate a restriction for HIV-2 EHO to produce core antigens in macaque PBMC and are consistent with the low infectious titer of HIV-2 EHO determined previously in macaque PBMC. On the other hand, infection of macaque and human PBMC with HIV-2/287 resulted in high levels of p27 production, at 1500 and 2000 ng/ml, respectively, confirming its ability to grow in cells of both species (Fig. 3).
Kinetics of reverse transcription
To identify the nature of the restriction for HIV-2 EHO in macaque cells, we analyzed the kinetics of reverse transcription in macaque and human PBMC infected by HIV-2 EHO and HIV-2/287 (Fig. 4). Early products of viral reverse transcription were detected with primers specific for the LTR R/U5 (Fig. 4a,b), and nearly completed proviral DNA was detected with primers specific for LTR U5/gag (Fig. 4c,d). Our results showed that early products of viral reverse transcription were synthesized during the first 4 h after infection of macaque PBMC by HIV-2 EHO and HIV-2/287 (Fig. 4a). With the same virus input (determined by p27 antigen), the levels of initiated viral DNA detected 4 h and 18 h post-infection were twofold higher for HIV-2 EHO than for HIV-2/287, indicating that, once HIV-2 EHO entered the macaque cells, it could initiate reverse transcription with the same efficiency as HIV-2/287. However, the level of initiated HIV-2/287 DNA increased markedly at 48 h when no further change was observed for HIV-2 EHO DNA. The accumulation of nearly completed proviral DNA observed for HIV-2 EHO 18 h after the infection of macaque PBMC (Fig. 4c) indicated that the virus was able to complete reverse transcription in macaque cells. The level of completed proviral DNA detected at 18 h post-infection is twofold higher for HIV-2 EHO than HIV-2/287, which correlated with the difference observed previously for the levels of newly initiated viral DNA (compare Fig. 4a and c). As seen for early viral DNA products, at 48 h after infection we observed a substantial increase of complete reverse transcription products for HIV-2/287, but not for HIV-2 EHO.
In parallel we analyzed the sequence of reverse transcription in human PBMC infected by HIV-2 EHO and HIV-2/287 (Fig. 4b,d). Our results showed that viral DNA synthesis was initiated within 4 h of infection of human PBMC with the two viruses (Fig. 4b), as was the case in macaque PBMC (Fig. 4a). The levels of newly initiated DNA detected at 4 and 18 h post-infection were significantly lower for HIV-2/287 than for HIV-2 EHO, consistent with HIV-2/287 being 100-fold less infectious than HIV-2 EHO. Because the infections were performed with the same quantity of p27 antigen input (50 ng/106 cells) for the two viruses, this corresponded to a multiplicity of infection of 1 for HIV-2 EHO and 10−2 for HIV-2/287 in human cells. Complete proviral HIV-2 EHO and HIV-2/287 DNA accumulated between 4 and 18 h post-infection (Fig. 4d), as seen previously for HIV-1 in stimulated PBMC . Consistent with the level of newly initiated products, we observed a significantly lower level of complete products for HIV-2/287 as compared with HIV-2 EHO at 18 h post-infection (Fig. 4d). A low level of proviral DNA was detected at time 0 (1 h post-adsorption) in HIV-2 EHO-infected human cells by the R/U5 primers (Fig. 4b), but not by the LTR/ gag primers (Fig. 4d). This was most probably due to partially reverse transcribed viral DNA in the resting virions .
The kinetics of reverse transcription for HIV-2/287 was similar in macaque and human PBMC (Fig. 4c,d). This is consistent with the observation that this virus attained similar titers (TCID50) and p27 production in these cells as shown previously (Fig. 3 and data not shown). In contrast, the level of HIV-2 EHO proviral DNA at 4 h post-infection was 40-fold lower in macaque PBMC than in human PBMC (Fig. 4a,b). These results indicated that HIV-2 EHO replication in macaque cells was blocked at an early stage, either at entry or at a point prior to the initiation of reverse transcription. This was not due to any limiting factor for virus replication in macaque cells, as the level of initiated viral DNA detected in HIV-2 EHO-infected macaque PBMC at 4 and 18 h post-infection was twofold higher than that observed in HIV-2/287-infected macaque PBMC (Fig. 4a).
To verify that the higher level of viral DNA detected previously for HIV-2 EHO in human PBMC was due to the 100-fold excess of infectious virus used (Fig. 4), we repeated the experiment using the same m.o.i. (rather than the same p27 antigen input) for HIV-2 EHO and HIV-2/287. Identical levels of initiated DNA products were detected at 18, 48 and 96 h post-infection in human PBMC infected by HIV-2 EHO and HIV-2/287 (data not shown). No signal was detected at 2 h post-infection for either of the two viruses, indicating that the initiated DNA products were synthesized between 2 and 4 h post-infection.
To demonstrate that HIV-2 EHO was capable of entering macaque PBMC and undergoing reverse transcription, we examined the effect of ZDV on the accumulation of RT intermediates in macaque and human PBMC after HIV-2 EHO or HIV-2/287 infection. ZDV treatment significantly reduced (by 70–92%) the accumulation of nearly completed proviral DNA in macaque and human PBMC infected by HIV-2 EHO and HIV-2/287 (data not shown), demonstrating that the majority of the proviral DNA detected resulted from reverse transcription after virus infection.
We demonstrate here that the adaptation of HIV-2 EHO to a highly pathogenic virus HIV-2/287 in M. nemestrina is not correlated with a change in coreceptor usage. Both viruses are able to use a number of coreceptors, but preferentially CXCR4 to infect human and macaque PBMC, consistent with recent studies suggesting that only CXCR4 or CCR5 are used by HIV in vivo [31,32]. Our results also indicate that CXCR4 usage per se is insufficient for enhanced virulence. However, it should be noted that our observations do not preclude the possibility that there might be quantitative differences between HIV-2/287 and EHO in their binding affinities for macaque CXCR4 receptor, or any other receptors not examined here.
Because HIV-2 EHO was originally isolated from an AIDS patient, its X4-usage is consistent with observations that X4-tropic viruses emerge late in the disease course in HIV-1 or HIV-2-infected patients [10,12,33,34]. This is in contrast with SIV isolates studied so far, which use predominantly CCR5 [15,35–37]. This contrast is of interest because of the genetic and structural homology between SIV and HIV-2, especially EHO, which belongs to the subtype B most closely related to SIVsm. Because HIV-2 is hypothesized to be the result of cross-species transmission of SIVsm, the acquisition of CXCR4 usage may have resulted from adaptation in humans. In addition, both HIV-2 EHO and HIV-2/287 are CD4-dependent viruses (data not shown), indicating that CD4-independent infection is not relevant for the adaptation of HIV-2 EHO in M. nemestrina.
Our study showed that HIV-2 EHO and HIV-2/287 present differential replicative capacity in macaque PBMC in vitro. The enhanced pathogenicity of HIV-2/287 in M. nemestrina correlates with its increased ability to replicate in macaque PBMC in vitro. These data are consistent with observations in SIV-infected macaques showing that late stage viruses are more aggressive in macaque T cells in vitro, but do not present any change in coreceptor usage [38–42]. Interestingly, the infectivity of HIV-2/287 is > 2 log10 lower than HIV-2 EHO in human PBMC (see Results, paragraph four), indicating that the adaptation of HIV-2 EHO in M. nemestrina is associated with a loss of replicative capacity in human cells.
Kinetic analysis of reverse transcription suggest that RT per se is not implicated in the restriction of HIV-2 EHO in macaque PBMC, as comparable levels of reverse transcription products were made in HIV-2/287 and HIV-2 EHO-infected cells (Fig. 4), similar to findings showing that RT is not involved in the restriction of HIV-1 in macaque PBMC . Instead, the low levels of proviral DNA initiated after HIV-2 EHO infection of macaque PBMC, as compared to EHO-infected human PBMC, indicate an early block for viral entry to macaque cells. Our experiments do not distinguish the early block being at entry or immediately afterwards. The facts that HIV-2 EHO and HIV-2/287 present the same CXCR4 usage and are CD4 dependent suggest that cellular determinants other than chemokine coreceptor or CD4 receptor are involved in this early restriction. At 48 h post-infection, a dramatic increase of proviral DNA was observed in HIV-2/287-infected cells (Fig. 4). This increase was probably due to second-round infections, as the addition of the peptide inhibitor TW70 6 h after the initial infection resulted in no change of proviral DNA levels at 20 and 48 h (data not shown). Therefore, overcoming an entry block of HIV-2 EHO could account for the enhanced growth of HIV-2/287 in macaque cells. However, we cannot exclude the possibility that additional block(s) exists in HIV-2 EHO-infected macaque cells such that reverse transcription products do not progress to form infectious virions. It is of interest to note that HIV-1 does not replicate efficiently M. nemestrina cells . Two levels of restriction have been suggested, one at an early phase of viral replication [45,46] and one beyond reverse transcription . Further analyses are necessary to confirm whether a second level of restriction exists for HIV-2 EHO in macaque PBMC.
In contrast to HIV-2 EHO, it has proven difficult to adapt HIV-1 by serial passages in M. nemestrina to produce a pathogenic virus capable of overcoming this restriction in vitro [47–50]. It remains to be determined whether this could be related to any hypothetical difference in the number of restrictions that have to be overcome by HIV-1 and HIV-2 EHO in order to replicate effectively in macaque cells.
Results presented here indicate that multiple interactions between viral and cellular factors, rather than chemokine coreceptor usage or reverse transcription per se, are involved in determining the efficiency of HIV-2 replication in macaque PBMC. Thus, HIV-2/287 infection of M. nemestrina provides a useful model to determine the viral and host determinants involved in the enhanced pathogenicity of HIV-2 in macaques and to study the role of immune response in the adaptation of the virus to a new host.
The authors thank E. Finn and J. Capalungan for technical support, V. Kewalramani, M. Emerman and A. Hovanessian for reagents, P. Firpo and M. Agy for helpful discussions, J. Overbaugh and M. Emerman for helpful critique, and A. Clarke for manuscript preparation.
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