Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) is the leading cause of HIV infection in infants. Direct infection of trophoblasts—cells forming the placental barrier—may cause this transmission. Entry of HIV-1 into trophoblasts is unusual for this retrovirus, because it is associated with endocytosis. However, given that trophoblasts express no or few receptors/coreceptors required for virus internalization, the mechanism underlying this event remains ambiguous. In the present study, we show that HIV-1 entry and infection of polarized trophoblasts are independent not only of CD4 but also of envelope (Env) glycoproteins gp120 and gp41. Virus internalization, cytoplasmic release, reverse transcription, integration, and HIV-1 gene expression occurred with both fusion-incompetent and Env-deficient viruses. Importantly, fusion-independent infection was observed when we used viruses produced in a natural cellular reservoir (i.e., primary human cells). Finally, HIV-1 requires heparan sulfate proteoglycans for uptake in trophoblasts. Together, our findings illustrate that HIV-1 utilizes an unusual pathway for entering human polarized trophoblasts.
More than 2 million children are living with HIV-1 worldwide, and 90% of these infections resulted from transmission from the mother  occurring in utero, at birth, or via breast-feeding . HIV-1 has been detected in newborns at delivery [3–5] and in aborted fetuses [6–9]. Importantly, women are becoming infected at a higher frequency than men , and access to antiretroviral treatment is limited in developing countries . Hence, definition of the mechanism(s) underlying vertical transmission is urgently needed.
For in utero HIV-1 transmission to occur, the virus must cross the selective placental barrier, which is composed of a double layer of polarized epithelial-type cells: cytotrophoblasts and syncytiotrophoblasts. The postulated models include cell-to-cell transfer, direct infection of the trophoblasts, and transcytosis of viral particles across trophoblasts (reviewed in ). The mechanism underlying infection with cell-free virus remains largely unknown. HIV-1 has been shown to productively infect trophoblasts both in vitro [13–16] and in vivo [5, 17–19]. However, they exhibit a much lower susceptibility to productive HIV-1 infection than do CD4= T cells [14, 15, 20].
HIV-1 entry in CD4= T cells is mediated by the virus-encoded envelope (Env) glycoproteins gp120 and gp41. These proteins interact with the primary cellular receptor CD4 and its coreceptors (e.g., CXCR4 or CCR5), which leads to the formation of a fusion pore in the host cell membrane (reviewed in ). Contrary to this, we have established that, on contact with human trophoblasts, HIV-1 is rapidly and massively endocytosed and virions are predominantly trapped within the endosomal compartments . It is noteworthy that this internalization event is crucial for HIV-1 infection to proceed in trophoblasts, given that drugs that inhibit the function of endosomes block virus infection [22, 23].
Although HIV-1 infection in trophoblasts relies on endo-cytosis, the initial events of the virus life cycle in this cell type are ill defined. For instance, the expression of CD4 remains very low to absent in primary trophoblasts, whereas the expression of coreceptors may vary during the course of pregnancy [15, 24, 25]. Some previous studies have addressed the contribution of CD4, CXCR4, and CCR5 in HIV-1 infection of trophoblasts, and the published reports are contradictory, either involving gp120/CD4/coreceptor interactions [20, 26] or not [16, 25, 27]. Hence, the role played by CD4 and coreceptors in HIV-1 entry into trophoblastic cells is still unclear, but it is reasonable to assume that CD4, CXCR4, and CCR5 are unlikely to play a significant role, because they are expressed at such low levels if present. In agreement with this, several groups have shown recently that CD4 is not required for HIV-1 infection in trophoblasts [27–30]. In this context, we developed 2 hypotheses. First, other attachment receptors must be involved to compensate for the low level of CD4/CXCR4/CCR5 in these cells. Second, if HIV-1 infection of trophoblasts is independent of the known receptor/coreceptors for entry, the virus may not rely on the Env glycoproteins for such an event. The central objective of the present work was to test the validity of these postulates.
Materials and Methods
Cells. Human trophoblastic cell lines (JAR and JEG-3), embryonic kidney 293T cells, the leukemic T cell line Jurkat (clone E6.1), and peripheral blood mononuclear cells (PBMCs) from healthy donors were maintained as described elsewhere .
Preparation of virus stocks. The NL4-3 (X4), JR-CSF (R5), NL4-3-Luc E−R+, HXB2-env (X4), Ada-M-env (R5), and HCMV-G molecular constructs used have been described elsewhere . The NL4-3-Luc E−R+ expression vector was used to generate Env-deficient reporter viruses (NL4-3ΔEnv). pHCMV-G codes for the broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G) . The fusion-competent pSM—wild type (WT) and fusion-incompetent pSM570R Env expression vectors were derived from the HXB-2 backbone . Viruses were produced by calcium phosphate transfection of 293T cells, as described elsewhere . Viruses loaded with the β-lactamase (βlaM)—Vpr fusion protein were produced by cotransfection of 293T cells with pCMV-βlaM-Vpr (provided by W. C. Greene, Gladstone Institute of Virology and Immunology, San Francisco, CA), together with some of the expression vectors described above. Virus preparations were also generated through acute infection of PBMCs with NL4-3 or JR-CSF.
Infection assays. Cells were incubated at 37°C for 24 h with NL4-3ΔEnv particles or recombinant luciferase-encoding viruses pseudotyped with SM-WT or SM-570R (1×106 target cells with 0–500 ng of p24 or, in some experiments, with 250 ng of p24 together with 10 μmol/L zidovudine [ZDV]). Virus gene expression was induced with tumor necrosis factor (TNF)— α (10 ng/mL), as described elsewhere . JAR cells were incubated for 30 min at 4°C with soluble CD4 (sCD4; 10 μg/ mL), an isotype-matched irrelevant control antibody (10 μg/ mL IgG2a), or a blocking anti-CD4 antibody (10 μg/mL SIM.2), after which Ada-M or HXB-2 pseudotyped reporter viruses (250 ng of p24) were added. Jurkat cells were exposed to the control antibody or SIM.2 for 30 min at 37°C before inoculation with pseudotyped viruses (100–150 ng of p24). T-20 (Roche Bioscience) was added at various doses (0, 0.2, 1.0, and 5.0 μg/ mL) at 4°C to JAR cells concomitantly with Ada-M or HXB-2 pseudotyped reporter viruses (250 ng of p24). Jurkat cells were treated with 1 μg/mL T-20 before exposure to HXB-2 pseudotypes or NL4-3 viruses (100–150 ng of p24). Luciferase activity was measured in lysed cells. For Jurkat cells infected with NL4-3, cell supernatants were harvested and frozen at −20°C until they were assayed using a homemade p24 test .
Virus absorption and internalization assays. Cells were exposed to pseudotyped SM-WT, pseudotyped SM-570R, NL4-3, or NL4-3ΔEnv (250 ng of p24 for 1×106 cells) for 0–4 h at 4°C (to test virus absorption) or 37°C (to test virus internalization). To test the kinetics of virus absorption, the cells were washed 3 times with ice-cold PBS. As for the kinetics of virus internalization, the cells were acid treated (0.5 mol/L NaCl and 1% acetic acid) for 1 min at 4°C and washed 3 times with ice-cold PBS to remove all uninternalized viral particles. Cells were lysed in ice-cold lysis buffer, as described elsewhere . Heparin sodium (0, 50, 250, and 500 IU/mL; Leo-Pharma) was added at 4°C together with NL4-3ΔEnv, Ada-M, or HXB-2 pseudotypes (250 ng of p24). JAR cells were pretreated with 0, 2, 20, 200, and 400 mU/mL heparinase (Sigma-Aldrich) for 1.5 h at 37°C.
βlaM-Vpr—mediated cleavage of CCF2/AM. The βlaMVpr assay was based on a technique established elsewhere [34, 35]. Briefly, polarized JAR cells were exposed to pseudotyped SM-570R or NL4-3ΔEnv viruses that were all loaded with βlaM-Vpr fusion protein (400 ng of p24 for 1×106 cells) for 2 h at 37°C. The virus-cell mixture was incubated with CCF2/ AM dye (Aurora Bioscience) for 1 h at room temperature, and the βlaM reaction was maintained for 24 h at room temperature in CO2-independent medium supplemented with 10% fetal calf serum (FCS) and 2.5 mmol/L probenecid (Sigma-Aldrich). Both the emission spectra of CCF2/AM (520 nm) and its cleaved product (447 nm), as well as the degree of βlaM-mediated cleavage, were assessed as described elsewhere .
Cell fractionation assays. Polarized JAR cells were exposed to pseudotyped (i.e., SM-WT, SM-570R, and VSV-G), NL4-3, or NL4-3ΔEnv viruses (250 ng of p24 for 1 × 106 cells) for 4 hat37°C. The cellular membranes were disrupted, and the 2 distinct fractions (i.e., cytosolic and endosomal) were isolated as described elsewhere .
Real-time polymerase chain reaction (RT-PCR). Polarized JAR cells ( 1 × 106) were either left untouched or were exposed to pseudotyped (i.e., SM-WT, SM-570R, and VSV-G) or NL43ΔEnv viruses (250 ng of p24) (a ratio of 1 × 106 cells to 100 ng of p24 was used for Jurkat) for 48 h at 37°C. For the experiment with the fusion inhibitor T-20, the drug was added (1.0 μg/mL) at 4°C to polarized JAR cells ( 1 X 106) concomitantly with NL4-3 or JR-CSF viruses produced in PBMCs (110 ng of p24). Total cellular DNA was isolated using the DNeasy Tissue Kit as recommended by the manufacturer (QIAGEN). To quantify the amount of integrated viral DNA, we used the RT-PCR approach described by Suzuki et al. .
HIV-1 entry and infection of human trophoblasts, independent of CD4 and gp120/gp41. Given that HIV-1 infection of trophoblasts relies heavily on the cellular endocytic machinery , maintaining cell polarity when studying such process is of prime importance. If the purification of primary human trophoblasts results in the loss of cell polarity , the human trophoblastic JAR cell line exhibits many characteristics of the early placenta . Importantly, these cells can be cultivated into a polarized epithelium-like monolayer that closely resembles the in vivo trophoblastic barrier .
Initially, treatment of polarized JAR cells with sCD4 had no effect on virus internalization (figure 1A). Our next series of experiments was aimed at defining whether agents that block gp120/CD4 interactions can affect HIV-1 infection. It should be noted that virus gene expression in such cells is detected only after the addition of physiologic doses of proinflammatory cytokines (TNF-α or interleukin-1α) . Pretreatment of polarized JAR cells with the blocking anti-CD4 antibody SIM.2 did not significantly modulate infection with R5 and X4 reporter viruses (figure 1B and 1C, respectively). As a control, infection of Jurkat T lymphoid cells with X4 pseudotyped reporter viruses was inhibited by 71% (figure 1D). Interestingly, virus gene expression was still efficiently inhibited in TNF-α— treated Jurkat cells (94% reduction). Expanding on these data, experiments were conducted with the fusion inhibitor T-20. The presence of up to 5 μg/mL T-20 did not reduce reporter gene activity of the 2 viral strains tested (figure 2A and 2B). In control studies, virus gene expression was inhibited by 76% in Jurkat cells in the absence of TNF-α and by 87% in its presence (figure 2C).
Next, polarized JAR cells were exposed to SM-570R pseudotypes or NL4-3ΔEnv viruses for increasing time periods at either 4°C (permits estimation of virus absorption) or 37°C (to estimate the kinetics of virus internalization). The SM-570R construct bears a mutation in gp41 that abolishes its fusogenic activity, whereas the NL4-3ΔEnv viruses completely lack Env because of a frameshift mutation near the env gene. The tested virus preparations were internalized with a high efficiency and in a timely fashion in polarized JAR cells (figure 3C and 3D). Importantly, they were also found to be internalized by JAR cells at a level comparable to that of WT viruses (figure 3A and 3B). We were intrigued by these data and tested whether virus infection could be observed independently of gp120 and gp41. Polarized JAR cells were exposed to increasing doses of reporter viruses pseudotyped with SM-WT Env and TNF-a was added to promote viral gene expression. As expected and in agreement with previous observations , we found that SM-WT viruses were able to infect the trophoblastic cell line JAR in a dosedependent fashion (figure 4A). The TNF-α—mediated reporter gene activity was increased by 37–46-fold, compared with unstimulated infected polarized JAR cells, which implied robust HIV-1 long terminal repeat (LTR)—driven gene expression. This infection was significantly diminished by the antiretroviral drug ZDV, which indicated that reverse transcription occurred in trophoblasts. Next, polarized JAR cells were exposed to SM- 570R pseudotyped and NL4-3ΔEnv viruses in escalating doses. Surprisingly, the Env-mutated and Env-deficient viruses were both found to infect JAR cells (figure 4B and 4C). Again, virus infection was markedly reduced by ZDV, illustrating true viral infection. Experiments performed with another choriocarcinoma cell line (i.e., JEG-3) confirmed that virus infection in trophoblastic cells can be achieved in a gp120/gp41-independent manner (figure 4D). However, no LTR-driven luciferase activity could be detected in the absence or presence of TNFa when Jurkat cells were inoculated with NL4-3ΔEnv virions in escalating doses (figure 4E).
Env-mutated and Env-deficient HIV-1 accessing the cytoplasm of polarized JAR cells. The distribution of HIV-1 within vesicular and cytosolic compartments was assessed in polarized JAR cells because productive HIV-1 infection has been reported to result from the release of p24 within the cytosol . VSV-G pseudotypes were used as positive controls for the assay because, although VSV-G pseudotypes enter cells through endocytosis, there is a rapid shift from the endocytic machinery to the cytoplasm . VSV-G pseudotypes were indeed predominantly located within the cytosolic fraction of JAR cells (figure 5A). Next, we found that 37% and 58% of the total internalized p24 content were located in the cytoplasm of JAR cells on infection with SM-WT pseudotyped and NL4-3 viruses, respectively. Importantly, fusion-incompetent and Env-deficient viruses were found to access the cytoplasm of polarized trophoblastic cells with efficiencies comparable to that of WT virus.
An alternative strategy was used to confirm the capacity of HIV-1 to gain access to the cytoplasm through a gp120/gp41- independent process. The fluorescence resonance energy transfer—based HIV-1 fusion assay exploits the incorporation of βlaM-Vpr chimeric proteins inside progeny viruses and their subsequent release into the cytoplasm of target cells as a marker of fusion. Infection with SM-570R and NL4-3ΔEnv viruses resulted in βlaM-mediated cleavages ranging from 4.60% to 19.33% and 7.98% to 11.70%, respectively (table 1). It is important to specify that, although HIV-1 is internalized primarily by endocytosis in trophoblasts , CCF2-AM is cytoplasmic and does not reach these compartments . Therefore, in trophoblasts, βlaM-mediated cleavage of CCF2-AM occurs when HIV-1 is gaining access to the cytoplasm after escaping the endosomal compartments.
Fusion-incompetent and Env-deficient viruses integrating within the genome of polarized trophoblasts. The presence of integrated viral DNA was next assessed in polarized trophoblastic cells after infection with fusion-incompetent and Env-deficient viruses by use of a RT-PCR test described elsewhere . As expected, we found that the number of integrated viral DNA copies obtained on infection with VSV-G pseudotypes was much higher than when WT HIV-1 was used (figure 5B). High quantities of integrated HIV-1 DNA copies were detected in trophoblasts on infection with SM-WT, in agreement with the notion of true HIV-1 infection in these cells. Importantly, the number of viral DNA copies obtained on infection with SM-570R and NL4-3ΔEnv viruses was comparable to the number observed when WT NL4-3 particles were used. We next tested viral integration using Jurkat cells as controls. Although integrated provirus was observed on infection of Jurkat cells with SM-WT, none was detected on infection with SM-570R or NL4-3ΔEnv viruses (data not shown).
The experiments conducted so far were done using viral preparations produced in human embryonic kidney 293T cells. Is the Env-independent virus infection of trophoblasts related to unique fusogenic properties derived from 293T cells (virus-producer cells) that would be in turn be acquired by HIV-1 on budding, or could this phenomenon be also seen when using viruses produced in other physiological cell types? Fully competent R5 (i.e., JR-FL) and X4 (i.e., NL4-3) viruses were harvested after infection of PBMCs and were used to infect trophoblasts in the presence or absence of T-20 before assessment of the amount of integrated viral DNA copies. We found that T-20 had no impact on the number of integrated viral DNA copies on infection of trophoblasts with HIV-1 produced in PBMCs (figure 5C). In a control experiment, integration of NL4-3 particles in Jurkat cells was reduced by 75% after treatment with T-20 (data not shown).
HIV-1 internalization in polarized trophoblasts, mediated at least partly through heparan sulfate proteoglycans (HSPGs). HSPGs have been suggested to be putative HIV-1 attachment/ entry receptors on the basis of the demonstration that heparinase treatment, which removes all cell-surface heparan sulfate chains, diminishes HIV-1 infection of HeLa-CD4 epithelial cells and monocyte-derived macrophages [43, 44]. Heparin strongly decreased entry of the 2 HIV-1 strains tested (figure 6A and 6C). To verify whether the effect induced by heparin was indeed related to receptor inhibition and not due to a steric hindrance phenomenon related to heparin, JAR cells were treated with heparinase to remove heparan sulfate from the cell surface before the cells were exposed to Ada-M or HXB-2 pseudotypes. A decrease in virus internalization was observed after treatment with heparinase (figure 6B and 6D). Moreover, internalization of NL4-3ΔEnv viruses was also diminished after pretreatment either with heparin (figure 6E) or heparinase (figure 6F). To define whether this diminution in viral internalization translated into reduced viral infection, infection assays were conducted in JAR cells pretreated with heparin. This treatment induced a 50% reduction in HIV-1 infection (figure 6G).
The present article addresses key aspects of the HIV-1 replicative cycle in polarized human trophoblasts.We used different technical strategies to demonstrate that the process of virus infection proceeds through a gp120/CD4-independent mechanism: (1) a blocking anti-CD4 antibody and sCD4, (2) the fusion inhibitor T-20, (3) fusion-incompetent viruses, and (4) viral particles lacking both gp120 and gp41 Env glycoproteins. Given that the cytoplasmic delivery of viral material, revere transcription (controlled by ZDV), and viral DNA integration are potent markers of true HIV-1 infection in CD4+ T cells and considering that these various events are observed in polarized trophoblasts after exposure to Env-deficient viruses at levels comparable to WT virions, we conclude that the process of HIV-1 internalization leading to the infection of polarized trophoblastic cells is independent of the normal interactions between CD4/coreceptors and gp120. Importantly, integration was seen in the presence of T-20 when fully competent viruses produced in natural cellular reservoirs, such as PBMCs, were used. This fact brings great credibility to the biological relevance of our findings. It is of interest to note that gp120-independent infection of CD4− epithelial cells and CD4+ T cells by HIV-1 has been documented previously . Moreover, although our experiments were performed with cell-free viruses, it has been previously proposed that HIV-1 infection of trophoblastic cells occurs mainly via cell-to-cell contact [27, 46]. The explanation for this discrepancy is presently unknown but could be related to differences in experimental methodologies, such as the use of polarized trophoblastic cells in the present work.
We also provide evidence that HSPGs play an important role in mediating Env-independent internalization of HIV-1 within trophoblasts. Heparan sulfate within HSPGs is linear, polysulfated, and, thereby, highly negatively charged glycosaminoglycan polysaccharides, and its binding to a wide variety of ligands is largely dependent on ionic interactions . This notion fully agrees with the observation that, although HIV-1 internalization is extremely rapid, binding of HIV-1 to trophoblasts is weak . In support of the idea that HSPGs may be involved in HIV-1 trophoblast infection in vivo, syndecan- 1 is expressed throughout pregnancy by syncytiotrophoblasts, whereas syndecan-2, syndecan-4, and glypican-1 are expressed by villous and extravillous cytotrophoblasts as well as by syncytiotrophoblasts . Neither heparin nor heparinase completely blocked viral uptake in trophoblasts, which suggests that other types of receptors may act concomitantly for HIV-1 internalization in trophoblasts. It has been shown that transcytosis of HIV-1 across the trophoblatic cell line BeWo can be blocked by antibodies to galactosyl ceramide . In addition, several putative alternate HIV-1 receptors, which are recognized to act as such on other cell types, are present on the surface of trophoblasts (including C-type lectins, such as the mannose receptor). It has also been hypothesized that vertical transmission of HIV-1 may be favored by the presence of dendritic cell— specific intercellular adhesion molecule 3—grabbing nonintegrin (DC-SIGN)/DC-SIGN—related—expressing cells within the placental environment . However, this property would apply to transmission via microbreaches, because these attachment receptors are expressed in maternal macrophages, placental capillaries, and Hofbauer cells but not in trophoblasts .
We have previously shown that endosome inhibitors abolish HIV-1 infection of trophoblastic cells [22, 23], which implies that the process of virus infection is endocytic in nature. On the basis of our present data, we conclude that HIV-1 endosomal escape occurs in trophoblastic cells through a mechanism that is independent of the viral Env glycoproteins. The precise mechanism through which HIV-1 can reach the cytoplasm and integrate within the host chromatin of polarized trophoblasts is still undefined. However, it is well documented that HIV-1 carries a lipid membrane and acquires a wide array of host cell proteins upon budding (reviewed in ). HIV-1 may have evolved to exploit the endosomal hydrolases to escape into the cytoplasm. However, this hypothesis is not supported by some recent data . Alternatively, HIV-1 might undergo fusion within the endosomes via interactions between virus-anchored host proteins and endosomal proteins within trophoblasts. Interestingly, it was recently shown that the nucleocapsid of VSV is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes . Moreover, it is recognized that HIV-1 infection from the endosomes is possible in macrophages, lymphocytic cells, and HeLa cells when viral particles are spared from degradation [52, 53]. Finally, images of HIV-1 particles internalized in endocytic vesicles and undergoing fusion with endosomal membrane have been observed in macrophages and trophoblastic cells [16, 54].
In summary, the data presented in this study further support the idea that HIV-1 infection of trophoblasts is fundamentally different from what is known for CD4+ T cells. Our model proposes that the initial interactions between HIV-1 and polarized trophoblasts occur partly through a HSPG-mediated absorption mechanism. This process leads to a rapid and massive endocytosis of virions. Infection ensues in the absence of the viral Env glycoproteins gp120 and gp41 through endosomal escape and access to the cytoplasm. Collectively, these data provide key information about the early events associated with the HIV-1 life cycle in polarized human trophoblasts.
We thank Sylvie Meéthot for editorial assistance.