Redox-Triggered Infection by Disulfide-Shackled Human Immunodeficiency Virus Type 1 Pseudovirions

MAbs, peptides, and sera.

The following anti-gp120 MAbs were used (each a whole immunoglobulin G [IgG], unless specified): CD4 binding site-overlapping (CD4bs) MAb IgG1b12 and its monovalent fragment, Fab b12 (5); CD4-IgG2, a chimera containing four copies of CD4 domains 1 and 2 fused to a IgG Fc domain (35); 2G12, against a unique gp120 epitope formed by terminal residues of N-linked glycans (41, 44); MAb 17b and Fab X5, directed to CD4-induced (CD4i) epitopes (33, 45); and 447-52D, against the V3 loop (9). MAbs against gp41 included 2F5 and 4E10, against a C-terminal region of the gp41 ectodomain (34, 52); 7B2, against the gp41 cluster I region; and 2.2B, against the gp41 cluster II region (3). MAbs 2F5, 4E10, and 2G12 were provided by H. Katinger (Polymun Scientific Inc., Vienna, Austria). MAbs 17b, 7B2, and 2.2B were provided by J. Robinson (Tulane University, Tulane, La.). MAb 447-52D was provided by S. Zolla-Pazner (Veterans Affairs Medical Center, New York, N.Y.) and the AIDS reference reagent program. MAbs IgG1b12, 2G12, 2F5, 4E10, and the CD4-IgG2 chimera (PRO 542) are broadly neutralizing (5, 34, 35, 44, 52). MAb 17b neutralizes diverse strains of HIV-1 in the presence of recombinant soluble CD4 (sCD4) (45). Fab X5 is also broadly neutralizing (33). Purified HIV immune globulin (HIVIG) was obtained from J. Mascola (Vaccine Research Center, National Institutes of Health, Washington, D.C.). Potently neutralizing serum from the HIV-1-infected donor FDA2 (40) was provided by G. Quinnan (Uniformed Services University of the Health Sciences, Bethesda, Md.). Other HIV-1-positive sera were derived from donors DA, CM, MT, and AC (1a).

The T-20 peptide (49) is based on a sequence from the gp41 C-terminal helical region and was synthesized and handled as described previously (35). PA14 (PRO140) is a mouse MAb to CCR5 (47).

Plasmids and mutagenesis.

The plasmid pCAGGS (36) was used to express membrane-bound Env of the primary R5 isolate JR-FL. We expressed Env proteins as both full-length gp160 and a mutant, truncated at residue 708, leaving three amino acids of the gp41 cytoplasmic tail, termed ΔCT. Mutations were made to introduce cysteines at residues 501 and 605 (the SOS mutant [3]). A mutation was generated to replace the gp120/gp41 cleavage site REKR by the inefficiently cleaved GEKR, termed UNC. Plasmid pNL4-3.Luc.R-E-, expresses an HIV-1 genome fragment with frameshifts in Env and Vpr and a luciferase reporter gene in place of Nef (10).

Production of pseudovirions and viral infectivity assays.

Pseudoviruses were produced as described previously by transfection of 293T cells with pNL4-3.Luc.R-E- and Env-expressing pCAGGS-based plasmids (10). Single-round infections were performed using U87.CD4.CCR5, HOS.CD4.CCR5, Cf2Th.CD4.CCR5, or Cf2Th.synCCR5 cells (10, 28). Briefly, virus (normalized for p24 content by enzyme-linked immunosorbent assay) was incubated with cells for 2 h. In some experiments, 5 mM dithiothreitol (DTT) was subsequently added for 10 min. Luciferase activity was measured as described previously (10). All assays were performed at least in duplicate and were repeated for a total of at least four replicates to ensure consistency.

In assays where virus was pretreated with DTT, virus was subsequently pelleted in Eppendorf tubes at 21,000 × g for 30 min to remove DTT and then resuspended in fresh medium before proceeding with infection. In general, to normalize for the possible side effects of pelleting on virus infectivity, untreated virus was spun down in parallel and used as a control.

Neutralization assays.

Neutralization was measured in various formats with all incubations at 37°C, unless specified.

(i) Standard neutralization assay.

The SOS virus was incubated with inhibitor for 1 h before being transferred to U87.CD4.CCR5 cells for a further 2 h of incubation. Unbound virus was removed by changing medium, and the culture was incubated for a further 1 h. Cells were subsequently treated with 5 mM DTT for 10 min, and the medium was then replaced. Wild-type (WT) virus was used in neutralization assays using the same procedure and washes but without the DTT incubation. In a modified assay, the wash step was omitted and the inhibitor remained present until the DTT was removed. This is referred to as the standard (no wash) format.

(ii) Preattachment neutralization assay.

As in the standard assay, virus and inhibitor were incubated together for 1 h, but inhibitor was then removed by centrifugation in Eppendorf tubes for 30 min at 21,000 × g and resuspension of the virus pellet in fresh medium. Virus was then added to cells, and the assay was then performed in the same manner as the standard assay. In parallel controls, untreated virus was pelleted as above but before the 1-h incubation of virus with inhibitor and subsequent addition to cells.

(iii) Postattachment neutralization assay.

SOS virus was allowed to attach to cells for 2 h to form an SOS-arrested intermediate (SAI), and medium was replaced to remove any unattached virus. Inhibitors were then added for a further 1 h before DTT treatment (without replacing the medium) for 10 min. In a modified postattachment neutralization protocol, medium was replaced before as well as after the DTT incubation. This was referred to as the postattachment (washout) format.

To generate a temperature-arrested intermediate (TAI) for postattachment neutralization, WT gp160-bearing pseudovirions were allowed to attach to target cells at 14°C for 2 h. The medium was then replaced, and inhibitors were added for a further 1 h at 14°C before washing with phosphate-buffered saline, replacement of medium, and warming to 37°C.

Infectivity of WT and mutant pseudovirions.

We first examined the infectivity of pseudovirions bearing WT Env, SOS disulfide mutant Env (3), and proteolytically unprocessed Env (UNC). As expected, UNC pseudovirions were essentially noninfectious, since Env processing is required for fusion (30) (Fig. 1A). SOS virus was also noninfectious (Fig. 1A), presumably because the gp120-gp41 disulfide bond prevents fusion. Virions bearing WT gp160 with a truncated cytoplasmic tail, ΔCT, infected cells to the same extent as those bearing full-length gp160 (Fig. 1A). It has been previously shown that cytoplasmic tail truncation increases the exposure of the CD4 and coreceptor binding sites, thus rendering the virus more sensitive to neutralization (17). Since this property might allow a somewhat greater sensitivity to titrate the effects of fusion inhibitors, ΔCT Env-bearing viruses were selected for further experiments.

Fusion of the SOS-arrested intermediate by addition of reducing agent.

In a cell-cell fusion assay, SOS fusion was found to be DTT dependent (1). Building on this result, we determined whether infection of SOS pseudovirions could be rescued by reduction of the SOS disulfide bond. Thus, target cells preincubated with SOS pseudovirions were treated with 5 mM DTT. This resulted in infection of SOS virus, similar to WT levels (Fig. 1B), but did not affect the WT or UNC pseudoviruses. Further experiments confirmed 5 mM as the optimal DTT concentration, and this was used subsequently (Fig. 1C).

Reducing agent induces fusion only after virus attachment.

We next examined whether the timing of DTT treatment is important for inducing SOS fusion. When free pseudovirus was pretreated with DTT and then pelleted, resuspended in fresh medium, and added to cells, infection remained inefficient (Fig. 1D). However, DTT treatment of virus both before and after attachment to cells gave results identical to those obtained with DTT posttreatment alone. Thus, DTT pretreatment does not affect virus infectivity. Overall, DTT treatment after virus attachment to cells was necessary and sufficient for SOS fusion (Fig. 1D). Therefore, infection proceeds via a novel, redox-sensitive structure. We refer to this as the SAI. We next explored the nature of the SAI by examining its susceptibility to various classes of HIV-1 entry inhibitors.

The SAI engages both CD4 and coreceptor.

To determine if SOS pseudovirions attach to the CCR5 coreceptor, we used an anti-CCR5 MAb (PA14) that is known to inhibit infection (47). PA14 effectively inhibited SOS infection when added before or concurrently with virus (Fig. 2A). However, if added after the SAI had formed, it had no effect (Fig. 2A). A similar result was found using the anti-CCR5 MAb 2D7 (1). These results suggest that the SAI engages both CD4 and CCR5, but they do not exclude the possibility that virus attachment sterically obstructs anti-CCR5 MAb binding. To further explore this issue, virions were preincubated with 10 μg of sCD4/ml and then incubated with CCR5⁺ CD4⁻ cells. Infection was observed in the presence but not absence of sCD4, suggesting that the virus binds to CCR5 in a CD4-dependent manner (Fig. 2B). Consistent with this notion, prior studies demonstrated that sCD4 induces exposure of the coreceptor binding site on soluble SOS Env proteins (3).

The SAI is sensitive to entry inhibitors that target gp41.

Based on the above findings, SOS pseudovirions offer a unique opportunity to dissect the mechanisms of viral entry inhibitors at physiologic temperature. In turn, this might allow further characterization of the nature of the SAI. Three neutralization assays were developed (see Materials and Methods). In the standard neutralization assay, virus and inhibitors are preincubated prior to their addition to cells. In the preattachment neutralization assay, inhibitors are incubated with virus and then removed by centrifuging virus. In the postattachment neutralization assay, inhibitors are added after the SAI has formed but prior to the addition of reducing agent.

Initially, we compared WT and SOS pseudoviruses in the standard neutralization assay. WT and SOS viruses showed similar sensitivities to the anti-gp120 MAbs IgG1b12 (5) and 2G12 (41, 44) and to the tetrameric CD4-IgG2 molecule (35) (Table 1). However, the SOS virus was neutralized more strongly by anti-gp41 MAbs 2F5 and 4E10 and the V3 loop-specific MAb 447-52D and more weakly by the T-20 peptide (Table 1).

We next compared the efficacy of various reagents to neutralize the SOS virus in standard and postattachment formats. IgG1b12 was active in both standard (Fig. 3; Table 1) and preattachment (Fig. 4A) assays. However, postattachment neutralization was much weaker, consistent with the notion that virus-cell attachment blocks MAb access. In contrast to findings in a previous report, Fab b12 also did not exhibit significant postattachment activity (31) (Table 1). Similarly, MAbs 447-52D and 2G12 did not effectively neutralize postattachment. The lack of postattachment neutralization by the V3 loop-specific antibody 447-52D can be explained by the involvement of the V3 loop in coreceptor engagement (11). We propose that 2G12 binding may be sterically occluded in the SAI or else it may bind to the complex, but it can no longer inhibit fusion. CD4i MAbs that overlap the CCR5 binding site (46, 50) neutralized weakly in all formats (Fig. 3l Table 1). Thus, the postattachment potency of 17b and Fab X5 was similar to that of MAb IgG1b12 in the same assay format (Fig. 3; Table 1). In contrast, 2F5 and another neutralizing anti-gp41 MAb, 4E10, neutralized effectively in the postattachment format (Fig. 3; Table 1). Furthermore, 2F5 performed relatively poorly in a preattachment neutralization assay (Fig. 4A). Previous studies indicated that although MAb 2F5 potently neutralizes HIV-1, it does not bind well to native Env (43). It is possible, then, that 2F5 neutralizes virus after it attaches to cells (42). As expected, nonneutralizing anti-gp41 MAbs 7B2 and 2.2B had no activity in either neutralization assay format (Fig. 3 and data not shown).

The peptide T-20 (22) was the only reagent to perform better in the postattachment format than the standard format (Fig. 3, Table 1). In agreement with this, it performed poorly in the preattachment neutralization assay (Fig. 4A). Compared with MAb 2F5, T-20 activity was more sensitive to increased wash steps (Fig. 4B). Remarkably, T-20 appears to exert its potent inhibitory effect largely during the short DTT incubation period, suggesting that fusion proceeds rapidly upon triggering. T-20 might bind to virus more effectively (and perhaps irreversibly) after the fusion potential of the SAI is released, in agreement with separate studies (1).

Comparison of the SAI and temperature-arrested fusion intermediates.

Traditionally, fusion intermediates have been studied in the form of TAIs (24, 32). To compare these to the SAI, we produced a full-length gp160 WT TAI at 14°C. The TAI was inhibited more potently by the T-20 peptide but more weakly by 2F5 and not at all by IgG1b12 (Table 1). This indicates that the TAI had progressed further towards full fusion by the time the inhibitors were added. The decreased 2F5 activity can be partially explained by the low temperature, since 2F5 neutralization of SOS virus was also weaker in these conditions (data not shown). In contrast to the TAI described here, a WT TAI (1) produced at 18°C appeared to be arrested at an earlier stage in the fusion cascade than the SAI. We do not have a direct explanation, but the difference probably stems from the use of different cell lines and the precise assay details. Certainly, this argues that while the SAI behaves quite predictably in different assays, TAIs appear to be somewhat less predictable.

Antibodies capable of postattachment neutralization constitute a significant fraction of the total anti-Env antibody in HIV-positive sera.

We showed above that the CD4i MAbs X5 and 17b and the gp41 MAbs 2F5 and 4E10 are able to neutralize virus in the postattachment format. Conversely, MAbs IgG1b12, CD4-IgG2, 447-52D, and 2G12 neutralize at least 1,000-fold less efficiently in the postattachment format than in the standard neutralization format. Although not proven, it is generally considered that CD4bs and V3 loop antibodies constitute a dominant fraction of the neutralizing activity in HIV-positive sera and antibodies like 2F5 and 4E10 are extremely rare (15, 52). We sought to reevaluate the strength of the postattachment neutralizing antibody component of HIV-positive sera and HIVIG using the SAI. Surprisingly, HIV-positive sera neutralized SOS virus only 4- to 38-fold more weakly (mean = 19) in the postattachment format than in the standard format (Fig. 5). This stands in stark contrast to MAb IgG1b12, which neutralizes approximately 1,000-fold more weakly in the postattachment format and more closely mimics the behavior of MAb 2F5, which neutralizes only about 10-fold less effectively in this format. This result suggests that the neutralizing activity of the HIV-positive sera does not derive solely from V3 loop and CD4bs antibodies. The sera appear to contain a significant component of 2F5-like or 17b-like antibodies, or antibodies to as yet uncharacterized epitopes that are able to mediate postattachment neutralization. In fact, several MAbs to the 2F5 and CD4i epitope clusters have been isolated from a phage-displayed antibody library prepared from the FDA2 donor (33, 52).

The above findings provide a means of estimating concentrations of particular MAbs in sera that would be needed to obtain the postattachment neutralization observed. Postattachment 50% inhibitory concentration (IC₅₀) of sera is, on average, approximately a 1:26 serum dilution (Fig. 5). Postattachment IC₅₀ of 17b is approximately 30 μg/ml. If we assume that serum postattachment neutralization is comprised entirely of neutralizing antibodies similar to 17b, then 780 μg/ml would be required. Since the total concentration of anti-Env antibody has been previously estimated at between 100 μg/ml and 1 mg/ml (2), it appears unlikely that such a large proportion of anti-Env antibody would recognize the CD4i epitope (78 to 100%). However, it should be noted that the JR-FL isolate is particularly resistant to CD4i MAbs and that other CD4i antibodies may be more potent than 17b (33). Using a similar calculation, serum postattachment neutralization is equivalent to 7.8 μg of 2F5/ml or 78 μg of 4E10/ml. This translates as 0.78 to 7.8% of the total anti-Env fraction of sera for 2F5 or 7.8 to 78% for 4E10.