Characterization of a Late Entry Event in the Replication Cycle of Human Immunodeficiency Virus Type 2

Cells. (i) PBMCs.

Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats. Buffy coats were diluted in an equal volume of RPMI 1640 (Gibco), layered onto Ficoll Hypaque (Pharmacia) and centrifuged at 800 × g for 30 min at room temp. The white cell layer was harvested, washed in medium, and cultured at 10⁶ cells/ml of RPMI 1640 medium supplemented with 20% fetal calf serum (FCS), 60 μg of penicillin/ml, and 100 μg of streptomycin/ml. Cultures were stimulated for 2 to 3 days with phytohemagglutinin (PHA; 0.5 μg/ml) and then cultured with interleukin-2 (IL-2; 20 U/ml) for 2 to 3 days prior to infection.

(ii) Primary macrophages.

Fresh blood (buffy coat) was diluted in an equal volume of phosphate-buffered saline (PBS) containing 10 mM d-glucose and 2.5 mM EDTA, layered onto 15 ml of Ficoll Hypaque (Pharmacia) in a 50-ml tube, and centrifuged at 2,000 rpm for 30 min at room temperature. The PBMC interphase was carefully removed and washed once with PBS containing 10 mM d-glucose and 2.5 mM EDTA before centrifugation at 350 × g for 7 min. The supernatant was removed, and cells washed once more with RPMI 1640 before resuspension in RPMI 1640 containing 5% FCS. Cells were counted, diluted to 10⁸/15 ml, and plated onto a 14-cm-diameter bacterial petri dish. Cells were incubated for 2 h at 37°C. The adherent cells were then gently washed, first with the medium on the plate and then twice with fresh RPMI 1640–5% FCS, before 15 ml of RPMI 1640 containing 10% human serum was added, and cells were incubated at 37°C overnight. The following day, adherent cells were washed again and fresh medium was added. Macrophages were ready for infection 5 to 7 days later. The day prior to infection, the macrophages were gently scraped off the dish using a cell scraper. Cells were counted and reseeded into 48-well tissue culture trays (2 × 10⁴/well) or small flasks.

(iii) Cell lines.

T-cell lines C8166 and H9 were cultured in RPMI 1640 medium supplemented with 10% FCS and pen/strep. The human glioma cell lines U87/CD4 and U87/CD4/CXCR4 (12), the human osteosarcoma cell line HOS, GHOST and the GHOST-derived lines expressing CCR1, CCR2b, CCR3, CCR5, and CXCR4 (7), HeLa/CD4 (36), and Magi (27) derivatives were all cultured in Dulbecco's minimal Eagle's medium (DMEM) supplemented with 5% FCS, 60 μg of penicillin/ml, and 100 μg of streptomycin/ml.

Viruses.

The HIV-2 isolates used have been described elsewhere (39, 50). prCBL-23 virus was produced in PBMCs from HIV-negative donors. Stocks of TCLA CBL-23 were prepared in the CD4⁺ T-cell line C8166. ROD was generated from pACR23 (25) after transfection into H9 cells.

Infectivity assays.

Cells were seeded into 48-well trays on the day prior to infection, at 2 × 10⁴/well. Infections were performed in duplicate or with serial dilutions of 100 μl of cell-free virus supernatant. Virus was incubated with cell lines for 3 h before addition of 500 μl of growth medium. Infected cells were cultured for 3 to 4 days before being fixed in methanol-acetone and immunostained for viral antigens as described below (39). Supernatants (from nonadherent cells and primary macrophages) were assayed for RT activity by enzyme-linked immunosorbent assay (ELISA) (Retrosys RT activity kit; Cavidi Tech, Uppsala, Sweden).

Staining foci of infection.

Methanol-acetone-fixed cells infected with HIV-2 were immunostained using HIV-2-positive serum as described previously (39). β-Galactosidase conjugates of anti-human immunoglobulin G (IgG) (Southern Biotechnology Associates Inc.; dilution 1:400) were used to detect first-layer antibodies. Infected cells were immunostained blue by addition of 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal; 0.5 mg/ml in PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride) as previously described (10). Infected Magi cells were washed in PBS and fixed in 5% glutaraldehyde for 30 min, followed by one wash in PBS before addition of X-Gal substrate. Individual groups of blue-stained cells were regarded as foci of infection, and virus infectivity was estimated as focus-forming units (FFU) per milliliter of virus.

Preparation of VSV-G pseudotypes.

U87/CD4/CXCR4 cells were plated on 6-well trays at 5 × 10⁵/well 24 h prior to inoculation with 1 ml of prCBL-23 (4 × 10⁴ FFU). After 2 days infected cells were transfected with 10 μg of vesicular stomatitis virus envelope G protein (VSV-G) plasmid using a calcium phosphate system according to the manufacturer's instructions (Promega). Transfected cells were further incubated at 37°C for 3 days before the supernatant was harvested. The presence of pseudotype viruses that carried VSV-G glycoproteins was assessed on cells resistant to HIV-2 infection.

Preparation of mixed virion pseudotypes.

U87/CD4/CXCR4 cells (5 × 10⁵ per 25-cm flask) were coinfected with 5 × 10⁴ FFU of both prCBL-23 and ROD. Supernatants were harvested after 3 days.

RT ELISA.

The Lenti-RT activity assay (Cavidi Tech) was used for RT ELISA of supernatants obtained from infected PBMCs and macrophages according to the manufacturer's instructions. Briefly, the RT activity in the test sample synthesizes DNA and incorporates bromodeoxyuridine triphosphate (BrdUTP), which is detected and quantified by binding of an anti-BrdU antibody conjugated to alkaline phosphatase (AP). The AP activity is measured by addition of an appropriate substrate, and the color developed is proportional to the RT activity.

DOTAP treatment.

Serial dilutions of virus (100 μl) were preincubated with 100 μl of N[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) at 20 μg/ml (prepared in serum-free medium [Optimem; Gibco]) for 30 min at room temperature. _target cells (plated the previous day in 24-well trays at 2 × 10⁴/well) were washed twice with serum-free Optimem before addition of the virus-DOTAP mixture and incubation at 37°C for 1 h. The cells were then washed twice, 1 ml of 5% DMEM was added, and cells were incubated at 37°C for 3 days, after which they were fixed and stained.

Assays for viral entry and reverse transcription.

Virus was treated with DNase (50 U of DNase/ml plus 5 mM MgCl₂) for 1 h at 37°C. A 0.5-ml volume of treated virus (2.5 × 10³ FFU as assessed on U87/CD4/CXCR4 cells) was used to challenge _target cells: U87/CD4/CXCR4 cells, HeLa/CD4 cells, HOS/CD4/CXCR4 cells, PBMCs, and primary macrophages (2 × 10⁶ cells). Three controls for input DNA were included in these assays: (i) heat-inactivated virus (1 h at 60°C) added to cells, (ii) virus incubated on ice and washed with ice-cold medium three times, and (iii) untreated cells. Cells were washed three times, spun, and stored at −20°C for PCR analysis.

DNA was extracted from cultured cells using the DNA minispin kit from Qiagen according to the manufacturer's recommendations. PCRs were performed on a Robocycler (Stratagene) with the Expand Long Range PCR System (Roche Molecular) as recommended. The PCR for second-strand transfer products used forward primer 2713 (5′-TCTCTCCAGCACTAGCAGGTAGAG) and reverse primer 2715 (5′-CAAGACGGAGTTTCTCGCGCCCAT). Conditions were 40 cycles of 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, 30 s, with an initial denaturation at 94°C for 4 min prior to the first cycle. ERV-3 PCR was performed as described previously (20). PCR products were analyzed by electrophoresis through a 2% agarose gel stained with ethidium bromide (0.1 μg/ml).

A primary isolate of HIV-2 prCBL-23, but not TCLA CBL-23, replicates poorly in specific cells.

We previously observed that most primary isolates of HIV-2 could use CXCR4 as a coreceptor when expressed on U87 human glioma cells expressing human CD4 (U87/CD4). However, infection by some primary virus isolates was restricted in certain human cell lines (HeLa/CD4 and RD/CD4) that express high levels of both CD4 and endogenous CXCR4 (39). To investigate this restriction to infection further, we used the primary isolate prCBL-23 and a TCLA CBL-23. We tested the tropism of CBL-23 and prCBL-23 for HeLa/CD4, HOS/CD4/CXCR4, U87/CD4/CXCR4, and the CD4-expressing T-cell line C8166 as well as PBMCs and primary macrophages. Figure 1A shows that infection of HeLa/CD4 and HOS/CD4/CXCR4 cells is less efficient for both CBL-23 and prCBL-23 compared with infection of U87/CD4/CXCR4 cells. However, whereas CBL-23 is only 5- to 10-fold less efficient, prCBL-23 is 40- to 100-fold less efficient at infection of HeLa/CD4 and HOS/CD4/CXCR4 cells, respectively. The difference for CBL-23 compared to prCBL-23 was even more pronounced in primary macrophages. Figure 1B shows that prCBL-23 is not restricted in C8166 cells or in primary lymphocytes (although replication is 2 to 3 days slower in PBMC), whereas infection of primary macrophages is more than 100-fold less efficient for prCBL-23 than for CBL-23. The restriction, however, was incomplete, with some background infection in HeLa/CD4 and HOS/CD4/CXCR4 cells. This “background virus” in HeLa/CD4 cells was rescued into permissive cells (C8166), and the phenotype was shown to be that of prCBL-23 and not a low level of variant viruses with the CBL-23 phenotype (data not shown). The restriction to infection in HeLa/CD4 cells was confirmed in the Magi cell line, a derivative of HeLa/CD4 cells posessing a lacZ gene under the control of an HIV long terminal repeat (LTR) reporter (27). The lacZ gene is activated when Tat produced by integrated provirus binds to the LTR and drives expression of β-galactosidase. CBL-23 efficiently induced β-galactosidase expression, while prCBL-23 did not (data not shown).

The restriction cannot be overcome by expression of alternative coreceptors on HOS cells.

We previously reported that prCBL-23 can use a broad range of coreceptors in addition to CXCR4 including CCR1, CCR2b, CCR3, and CCR5 (39). We tested whether the restriction to infection of HOS/CD4/CXCR4 cells was active only when the CXCR4 coreceptor was used and whether the use of alternative receptors could relieve it. A panel of GHOST cells (7) (derivatives of HOS cells expressing one of the coreceptors CCR1, CCR2b, CCR3, CCR5, and CXCR4) was tested for the infectivity of prCBL-23. All of these GHOST cells expressing various coreceptors were fully susceptible to infection by CBL-23 but restrictive to infection by prCBL-23 (data not shown). Thus, the restriction of prCBL-23 cannot be overcome if the infection is directed via any of the coreceptors previously shown to be used by prCBL-23 (on U87/CD4 cells). Furthermore, the restriction is not confined to a single clone of HOS cells.

Postintegration events are not restricted in HOS and HeLa cell lines.

Treatment of retroviral vectors with the cationic liposome DOTAP enhances the rate of transduction of _target cells (44). We investigated whether DOTAP could overcome the restriction to infection of HOS and HeLa cells by treating virus with DOTAP for 1 h before challenging cells (CD4⁺/CXCR4⁺ HOS or HeLa). The results are shown in Fig. 2. DOTAP has little or no effect on the ability of CBL-23 to infect HOS or HeLa cells. However, the effect of DOTAP on the infection of HOS or HeLa cells by prCBL-23 was remarkable, enhancing infectivity at least 100-fold compared to that for untreated cells. The mechanism of the DOTAP enhancement is unknown, but this experiment shows that HOS and HeLa cells can accommodate integration and the production of viral proteins. However, it is possible that DOTAP simply enhanced background infection of variant viruses that had the same phenotype as TCLA CBL-23. We therefore rescued the progeny of DOTAP-enhanced virus from HOS/CD4/CXCR4 or HeLa/CD4 cells into permissive C8166 cells (to make high-titer stocks) and tested for infectivity of U87/CD4/CXCR4 and HOS/CXCR4 cells. The phenotype of the rescued virus was the same as the original prCBL-23 (data not shown). These results show that restrictive cells can produce fully infectious prCBL-23 virions provided the restriction to infection is bypassed by DOTAP treatment.

The envelope of prCBL-23 can function for cell-cell fusion.

We investigated whether the restriction to infection of HOS and HeLa cells is at the cell surface and is due to a defective prCBL-23 envelope. C8166 cells were infected with prCBL-23 or CBL-23 and 3 days later were cocultivated with CD4⁺/CXCR4⁺ HOS or HeLa cells as _target cells. After overnight incubation the adherent _target cells were stained and examined for the presence of syncytia. Figure 3 shows that, as expected, the nonrestricted CBL-23 can fuse with both _target cell types, but it also shows that the restricted prCBL-23 efficiently induced fusion with these cells. Thus, the restriction to infection is not at the cell surface and is not due to a defective prCBL-23 envelope.

prCBL-23 envelope can function for cell-free virus infection.

The above experiment demonstrates that there is no restriction to cell-to-cell fusion induced by the prCBL-23 envelope. However, the capacity of the prCBL-23 envelope to induce cell-to-cell fusion may not reflect the ability of cell-free viral particles to fuse with and enter cells (38). To address this question, we used mixed virions of prCBL-23 and the prototype strain of HIV-2, ROD. Supernatant from U87/CD4/CXCR4 cells coinfected with prCBL-23 and ROD was harvested to produce pseudotype virus (prCBL-23/ROD). Such pseudotypes have the envelopes of both prCBL-23 and ROD. Cells were challenged with the prCBL-23/ROD pseudotype in the presence of an anti-envelope neutralizing antibody to ROD (monoclonal antibody [MAb] 28.8e) (40) to block any entry mediated by the ROD envelope. At least 2 log units of infectivity of the ROD strain was neutralized in the presence of 28.8e, while there was little reduction of the prCBL-23/ROD pseudotype on either HOS/CD4/CXCR4 or HeLa/CD4 cells (Fig. 4). Since the anti-envelope neutralizing MAb 28.8e specifically neutralizes the ROD and not the prCBL-23, the infectivity of the pseudotype in Fig 4B is due to the prCBL 23 envelope. Thus, the envelope of prCBL-23 on cell-free virions can mediate infection of these cell types. Furthermore, the ROD strain of HIV-2 can overcome the restriction of HOS and HeLa cells to infection by prCBL-23. Since these pseudotypes will have all viral components mixed including cores, this experiment also shows that the prCBL-23 phenotype is not dominant.

PrCBL-23 can enter and reverse transcribe in the restrictive cell types.

To determine whether prCBL-23 can enter the restrictive cell types, we designed primers to detect the DNA transcripts produced at various times after entry into cells, including strong-stop DNA and DNA intermediates following the first (U3–U5) and second (LTR-gag) strand transfers. Equivalent doses of CBL-23 and prCBL-23 (measured on U87/CD4/CXCR4 cells) were treated with DNase and added to _target cells for 1 h before being washed and incubated for different periods up to 72 h. Cells were harvested, and DNA was prepared for PCR assays. As expected, we detected strong-stop DNA (not shown) and LTR-gag DNA in all three CD4⁺/CXCR4⁺ cell lines, U87, HOS, and HeLa, as well as in primary macrophages and PBMCs, after challenge with CBL-23 and prCBL-23 (Fig. 5). Importantly, similar levels of LTR-gag DNA were detected for prCBL-23 as for CBL-23 on all cell types, even though HOS and HeLa cells and primary macrophages are not fully permissive for infection with this isolate. These results demonstrate that prCBL-23 can enter HOS and HeLa cells and primary macrophages and that reverse transcription can be completed. These results distinguish the prCBL-23 restriction from the block to HIV replication noted for resting T-cells, where only incomplete reverse transcripts are made (64).

The LTR-gag DNA product detected after macrophage infection by prCBL-23 was, however, transient, with positive detection at 24 h but none after 48 h (Fig. 5B). Since the late-switch primers will also amplify the LTR-gag region from integrated proviral DNA, this result is consistent with the inability of prCBL-23 to integrate in primary macrophages.

The restriction to infection is overcome by VSV-G envelope.

So far we have shown that prCBL-23 can fuse, enter, and reverse transcribe in the restrictive cells. Experiments where DOTAP enhanced infection by prCBL-23 to levels comparable to those with CBL-23 show that postintegration steps are fully accommodated. And apart from the macrophages, the cells are actively dividing and a specific transport mechanism is not required because the PIC should have access to the nucleus during cellular division. We hypothesized that although the prCBL-23 PIC can be produced, it is held in an inappropriate compartment in the cell and may not have access to the nucleus even when the nuclear membrane is dissipated during cell division. We examined whether delivery of the viral core into the endocytic pathway would bypass the restriction in HOS and HeLa cells. VSV-G _targets HIV-1 entry into cells through an endocytic pathway (1, 35). A VSV-G(prCBL-23) pseudotype was prepared, and the restrictive cells were challenged (see Materials and Methods). Figure 6 shows that the incorporation of the VSV-G envelope into prCBL-23 results in enhanced infectious titers on HOS and HeLa cells to comparable levels to those of TCLA CBL-23. Thus the VSV-G envelope can impart to prCBL-23 the ability to infect previously restrictive cells, presumably by delivering the virus through the endocytic pathway.