The HR2 polymorphism N140I in the HIV-1 gp41 combined with the HR1 V38A mutation is associated with a less cytopathic phenotype
© Cunyat et al; licensee BioMed Central Ltd. 2012
Received: 9 November 2011
Accepted: 14 February 2012
Published: 14 February 2012
Resistance to the fusion inhibitor enfuvirtide (ENF) is achieved by changes in the gp41 subunit of the HIV envelope glycoprotein (Env). Specific ENF-associated mutational pathways correlate with immunological recovery, even after virological failure, suggesting that the acquisition of ENF resistance alters gp41 pathogenicity. To test this hypothesis, we have characterized the expression, fusion capability, induction of CD4+ T cell loss and single CD4+ T cell death of 48 gp41 proteins derived from three patients displaying different amino acids (N, T or I) at position 140 that developed a V38A mutation after ENF-based treatment.
In all cases, intra-patient comparison of Env isolated pre- or post-treatment showed comparable values of expression and fusogenic capacity. Furthermore, Env with either N or T at position 140 induced comparable losses of CD4+ T-cells, irrespective of the residue present at position 38. Conversely, Env acquiring the V38A mutation in a 140I background induced a significantly reduced loss of CD4+ T cells and lower single-cell death than did their baseline controls. No altered ability to induce single-cell death was observed in the other clones.
Overall, primary gp41 proteins with both V38A and N140I changes showed a reduced ability to induce single cell death and deplete CD4+ T cells, despite maintaining fusion activity. The specificity of this phenotype highlights the relevance of the genetic context to the cytopathic capacity of Env and the role of ENF-resistance mutations in modulating viral pathogenicity in vivo, further supporting the hypothesis that gp41 is a critical mediator of HIV pathogenesis.
HIV infection causes a progressive depletion of CD4+ T cells, which leads to the development of AIDS [1, 2]. Although CD4+ T cell loss in HIV infection is a multifaceted process [3–5], the death of bystander CD4+ T cells seems to be one of the main contributors to HIV-induced pathogenesis [6–8]. Various mechanisms have been proposed to explain the destruction of bystander CD4+ T cells, including apoptosis, autophagy or abortive infection [6, 8–11]. The HIV envelope (Env) glycoprotein, which mediates viral entry into the host cell by fusion of the viral and host cell membranes (reviewed in [12–14]), is one of the viral factors involved in the death of both infected  and bystander cells [7, 8, 16]. The Env complex is composed of two non-covalently linked subunits, namely, the surface glycoprotein (gp120) and the transmembrane glycoprotein (gp41), and is displayed as heterotrimers on the surface of virions and infected cells [14, 17–20]. Viral entry is a multistep phenomenon: the interaction of gp120 with the host cell surface CD4-receptor, and either CCR5 or CXCR4 coreceptor enables gp41 subunits to trigger hemifusion events, thereby leading to fusion. The HIV gp41 is a classic type 1 fusion protein that contains three domains: an ectodomain, a membrane-spanning domain, and a long intracytoplasmic segment. The ectodomain of gp41 consists of an N-terminal fusion peptide followed by two conserved coiled-coil domains that are referred to as C- and N-terminal heptad repeats (HR1 and HR2), which are connected by a non-helical loop region. These HR interact with each other in a leucine zipper-like fashion to mediate membrane fusion . Synthetic peptides that bind to one of the HR motifs interfere with their interaction and thus inhibit viral entry [22, 23].
Enfuvirtide (ENF, T-20) is the first peptide approved for clinical use in HIV salvage therapy. This drug is a 36-amino acid peptide that was designed based on the amino-acid sequence of the HR2 domain of the gp41 subunit. This peptide prevents the HR1-HR2 interaction by binding to the HR1 domain [22, 24, 25]. The therapeutic benefits of ENF therapy have been demonstrated by increases in CD4+ T cell counts and a significant reduction in HIV RNA levels [26–28]. Nevertheless, ENF-resistant HIV-1 variants rapidly emerge under drug pressure when virus replication is not completely suppressed [29–31]. Sequence analysis of ENF-resistant viral populations revealed the acquisition of mutations within the HR1 domain at positions 36-38 (GIV) [29, 30], which were associated with a reduction in viral infectivity, probably as a consequence of impaired interaction between HR1 and HR2 [32, 33]. However, certain compensatory mutations within HR2 may arise and restore viral infectivity [29, 32, 34–37]. Despite virological failure, specific mutations (the cluster V38A+N140I) have been associated with an increase in CD4+ T cell counts [38–40].
The Env glycoprotein plays a crucial role in the depletion of CD4+ T cells by inducing the death of single bystander cells, which is mediated by gp41 [41, 42]. Therefore, changes in gp41 that emerge under ENF pressure could induce a change in the viral pathogenicity. Although site-directed point mutations at position 38 in gp41 have been shown to exhibit deficiency in cell-to-cell fusion activity and apoptosis induction in vitro and in a humanized mouse model [43, 44], it is important to note that the genetic background has been proven relevant for functional evaluation of the ENF-resistant Envs because there may be compensatory changes that restore the infectivity of the virus [32, 34, 36, 37, 45, 46].
The objective of the current study was to evaluate the pathogenicity of several patient-derived gp41 proteins isolated from highly experienced patients receiving an ENF-containing salvage therapy and whether changes at position 38 and 140 in gp41 have an impact in the biological properties of patient-isolated Envs. Our results indicate that the primary gp41 Env proteins, with both V38A and N140I changes, induced lower levels of single-cell death and depletion of CD4+ T cells, although they retained cell-to-cell fusion activity. However, the mutation V38A in the context of a 140N or 140T change did not alter Env functions, underscoring the importance of the Env genetic background in the modulation of the cytopathic effects of the HIV-1 Env glycoproteins.
Results and discussion
Patients and envelope constructions
Characteristics of the three patients receiving an enfuvirtide-containing salvage therapy when samples were collected
No. of expression
Cell surface expression of HIV-1 recombinant envelope glycoproteins
Analysis of the Env protein fusogenicity
Quantification of envelope-induced absolute loss of CD4+T cells
Analysis of envelope-induced bystander apoptosis
The Env-mediated depletion of the CD4+ T cells may result from syncytium formation (directly related to fusogenicity values) or from gp41-mediated bystander apoptosis of single CD4+ T cells. Apoptosis is a crucial factor contributing to the loss of CD4+ T cells, and the extent of immune-cell apoptosis is correlated with AIDS progression . Importantly, apoptosis of bystander, uninfected cells is one of the major processes involved in the destruction of immune cells during HIV infection  because the majority of apoptotic CD4+ T cells in the peripheral blood and lymph nodes are uninfected in patients infected with HIV [57, 58]. The Env-induced death of single bystander cells is related to gp41-mediated membrane hemifusion processes between Env-expressing cells and target cells [41, 42]. This gp41 dependence suggests that drugs targeting gp41 function may alter HIV pathogenesis by inducing changes in the gp41 sequence. Consistent with this, certain ENF-resistant mutations arising during salvage therapy, specifically changes at position 38 in gp41, were associated with an immunological benefit, even after virological failure [39, 40]. Additionally, the increase in CD4+ T cells was enhanced by the concomitant polymorphism N140I . Because there were no significant differences in fusogenicity among the Envs tested in our assays, we specifically quantified Env-mediated cell death, since in vitro the single-amino-acid mutation V38A/E has been shown to alter this mechanism . HeLa cells expressing recombinant Envs were cocultured with primary CD4+ T cells. After 24 hours, the culture was stained with propidium iodide (PI) and DiOC6(3) to simultaneously determine the viability and the mitochondrial transmembrane potential of CD4+ T cells, respectively. Each Env was analysed in the absence and presence of the anti-CXCR4 antagonist JM-2987, and a correction for non-Env-mediated death was performed for each Env by subtracting the background death detected in the presence of JM-2987. When an intra-patient analysis of this assay was performed, there were differences between the recombinant clones carrying the V38A mutation and the wt clones in an N140I background, while the clones bearing the N or T amino acids at position 140 showed similar apoptosis-inducing capacity when wt and V38A clones were compared (Figure 4B). Thus, the impaired ability to deplete CD4+ T cells by the recombinant Envs that contained the cluster of mutations V38A+N140I in the gp41 protein was associated with a significant reduction in apoptosis induction on primary cells (13.4% and 7.4% for baseline and with the cluster of mutation samples, respectively; p = 0.031). In agreement with previously reported in vivo data , both the absolute loss of CD4+ T cells and Env-induced single cell death was significantly reduced for Envs that had the cluster of mutations V38A + N140I. However, the acquisition of the V38A mutation in a gp41 background containing an N or T at position 140 had no significant impact on the pathogenicity of Env, again highlighting the importance of the genetic context on the function of Env. Polymorphisms in HR2 have been shown to contribute to ENF resistance , and in this study, we demonstrate that they also have an important role in HIV pathogenesis. Nevertheless, a methodological limitation of our study is the relative small number of clones analyzed which gives us a low statistical power. A larger sample size would allow a more accurate estimation of the differences studied.
Recently, it has been reported that caveolin-1 modulates the Env-induced bystander apoptosis through interactions with gp41. None of the clones obtained from the patient carrying the N140I mutation in gp41 showed changes in the caveolin binding region, suggesting that the changes in the induction of bystander apoptosis observed in this study are not due to a defect in the binding of this protein to the gp41 protein.
Overall, the phenotype observed in the present study for Envs containing the cluster of mutations V38A+N140I, which maintained fusion capacity, but had a decreased ability to deplete CD4+ T cells, correlated with the observed in vivo data in patients, including a maintenance of viral load with an increase in CD4 counts. These results show that mutations that confer ENF resistance are associated with reduced pathogenicity in vivo.
In conclusion, these findings support the hypothesis that HIV gp41 is a critical mediator of HIV pathogenesis and suggest that it may be possible to target gp41 to attenuate HIV.
Three highly-experienced patients who were receiving an ENF-containing salvage therapy were selected from our previous study . These patients carried viruses that developed mutations at position 38 in the gp41 viral protein associated with drug resistance and had different changes at position 140. Two plasma samples from each patient were collected at baseline and during ENF treatment and were used to extract viral RNA.
Cell cultures and reagents
HeLa and TZM-bl cell lines were supplied by the NIH AIDS Research and Reference Program. The cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of heat-inactivated fetal calf serum (FCS) and maintained at 37°C in a 5% CO2 incubator. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from buffy coats obtained from a local blood bank (Banc de Sang i Teixits, BST), and CD4+ T cells were purified by negative immunomagnetic selection (Miltenyi Biotec, Spain). The final cell preparations were composed of > 95% CD4+ T cells as determined by flow cytometry. The isolated CD4+ T cells were incubated overnight at 37°C in RPMI media supplemented with 10% of FCS prior to use. All of the media were purchased from Invitrogen (Madrid, Spain).
The CXCR4 antagonist JM-2987 (hydrobromide salt of AMD-3100)  and the CCR5 antagonist TAK-779 [60, 61] were obtained through the NIH AIDS Research and Reference Program. The broadly gp120 neutralizing antibody 2G12 and the secondary antibody goat anti-Human IgG were obtained from Polymun (Vienna, Austria) and Jackson ImmunoResearch Laboratories (Pennsylvania, USA), respectively. The Tat expression plasmid pcTat was obtained through the NIH AIDS Research and Reference Reagent Program .
The cell tracker Dichloro-DimethylAcridin-One (DDAO) was purchased from Molecular Probes (Invitrogen, Madrid, Spain). The cationic fluorescent dye Propidium Iodide (PI) and the potentiometric mitochondrial probe DIOC6(3) were purchased from Sigma (Madrid, Spain) and Invitrogen, respectively.
The RNA from the plasma samples was isolated before and after the initiation of ENF treatment using the QIAmp Viral RNA kit (Qiagen). Full-length env/rev genes were amplified through RT-PCR using specific primers as previously described . A subsequent nested PCR was carried out using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) to obtain a fragment corresponding to the gp41 protein (primers MluF2 and RNANestedR corresponding to nucleotides 7726-7747 and 8882-8904 of the HIV HXB2 numbering system, respectively). A fragment corresponding to the gp120 protein was amplified from a NL4-3 plasmid (primers RNANestedF and MluR2 corresponding to nucleotides 5954-5983 and 7727-7747 of the HIV HXB2 numbering system, respectively). The purified gp41 and gp120 products, which overlapped each other in 22 bases, were combined by PCR and purified to obtain the recombinant Envs (gp120 from NL4-3 and gp41 from patients). A directional cloning reaction was performed to insert the fragment into the plasmid expression vector pcDNA.3.1D/V5/His-TOPO (Invitrogen), and several transformed bacterial colonies were selected for each sample. All recombinant plasmids were sequenced using specific primers, the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and an automatic DNA Sequencer (3100 Genetic Analyzer). The sequences were edited (using Sequencher, v4.7, from the Gene Codes Corporation, Ann Arbor, MI and GeneDoc, v2.6, software), and the recombinant plasmids with the required mutations were selected.
HeLa cells were plated at a density of 8 × 105 cells/well in six-well plates and allowed to grow overnight. The cells were transiently transfected (using Lipofectamine 2000 Reagent, Invitrogen, Spain) with 1.3 μg of the Env-expressing plasmids for the cocultures with primary cells or were cotransfected with the Env-expressing plasmids and 2.7 μg of pcTat for the fusion assays. Twenty-four hours post-transfection, the cells were collected for further analyses. As negative controls, cells were mock-transfected (with the pcDNA 3.1 vector) or transfected with pcTat alone.
Twenty-four hours post-transfection, cell membrane expression of the Env glycoprotein was assessed by flow cytometry after indirect staining with the anti-gp120 monoclonal antibody 2G12 (4 μg/ml) for 20 min at 37°C, followed by staining with phycoerythrin-labeled goat anti-human IgG (RT for 15 min). The cells were washed, fixed in 1% formaldehyde and analyzed by a FACS LSRII flow cytometer. The data were analyzed using FACSDiva software (BD Biosciences). Mock-transfected cells were used as a negative staining control. The percentage of Env-positive cells and the geometric mean fluorescence intensity (geoMFI) of these cells were considered as individual parameters or used to calculate the relative fluorescence intensity (RFI = % of Env-positive cells × geoMFI of Env-positive cells), as described previously .
Cell-to-Cell Fusion assays
Twenty-four hours post-transfection, Env/pcTat- and pcTat-transfected HeLa cells were cocultured with the reporter cell line CD4+/CCR5+/CXCR4+ TZM-bl for 6 hours in 96-well plates in the presence or absence of the CXCR4 and CCR5 co-receptor inhibitors JM-2987 and TAK-779 (1 μg/ml), respectively. The fusion efficiency of each clone was quantified by assessing the luminescence of the cells (Britelite kit, Perkin Elmer) with a Luminoskan Ascent luminometer (Labsystems, Spain).
Envelope-induced death in primary CD4+T cells: absolute cell loss and bystander apoptosis
Env-induced cytopathic effects were evaluated using a coculture system of Env-expressing HeLa cells as effector cells and labeled primary CD4+ T cells as target cells. The primary CD4+ T cells were stained with the far red cell tracker, DDAO (10 μg/mL), for 1 hour at 37°C. Env+ HeLa cells and CD4+/DDAO+ T cells were cocultured for 24 hours in the absence and presence of the inhibitor, JM-2987 (1 μg/mL), and were stained with DiOC6(3) (40 nM) and PI (5 μg/mL) for 1 hour at 37°C. Labeled microbeads (Beads Perfect Count, Invitrogen) were added to the stained coculture to quantify the absolute cell loss, and flow cytometry was performed by a FACS LSRII flow cytometer. The data were analyzed by the FACSDiva software (BD Biosciences).
The data were compared using non-parametric Mann-Whitney tests. All statistical analyses were performed using GraphPad Prism, version 5.01, for Windows (GraphPad Software, San Diego, California, USA). A P-value of 0.05 was considered to be significant for these studies.
This work was supported by the FIS project 07/0418 (to CC), the Spanish AIDS network, "RIS, Red Temática Cooperativa de Investigación en SIDA (RD06/0006)" and the CHAIN European Consortium. C. Cabrera and J. Blanco are researchers from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the Health Department of the Catalan Government (Generalitat de Catalunya). F. Cunyat is supported by the FIS project 07/0418 and VS is supported by grants from CHAIN, Collaborative HIV and Anti-HIV Drug-Resistance Network, Integrated Project no.223131, funded by the European-Commission Framework-7 Program. MC is supported by a RIS contract. This work is part of the PhD thesis of F. Cunyat at Universitat Autònoma de Barcelona, Barcelona, Spain.
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