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. Author manuscript; available in PMC: 2019 Oct 23.
Published in final edited form as: AIDS. 2018 Oct 23;32(16):2269–2278. doi: 10.1097/QAD.0000000000001953

Low levels of HIV-1 Envelope-mediated Fusion are Associated with Long-term Survival of an infected CCR5−/− Patient

Paul R GORRY 1, Fahim AHMAD 2, Jon MOHL 2, Ghalib ALKHATIB 3,*
PMCID: PMC6239422  NIHMSID: NIHMS980346  PMID: 30005022

Abstract

Objectives:

This study investigated whether Env-mediated fusion levels of R5X4 viruses are associated with long-term survival of an infected CCR5−/− patient.

Design:

Four R5X4 Envs were cloned from each of two infected homosexual individuals (DR and C2) homozygous for the CCR5Δ32 allele. DR is a long-term survivor chronically infected with HIV-1 and his Envs were cloned 12 years after testing HIV+, while C2 Envs were isolated 1 year after primary infection.

Methods:

The current study sequenced the gp41 subunits and created hybrid Envs that contained exchanged gp41 subunits or V3 loops. The Env-mediated fusion activity of Envs was examined in cell fusion and virus infection assays.

Results:

Sequence analysis indicated novel polymorphisms in the gp41 subunits of C2 and DR, and revealed sequence homology between DR and certain long-term non-progressors (LTNPs). The DR Envs consistently showed lower Env-mediated fusion, smaller size and delayed onset of syncytia formation. Envs containing swapped gp41 regions resulted in the transfer of most of the fusion phenotype and in the shift of the inhibition concentration 50 (IC50) of the inhibitory T20 peptide. In contrast, Envs with swapped V3 domains resulted in the partial transfer of the fusion phenotype and no significant change in the IC50 of T20.

Conclusions:

Env sequence polymorphisms identified two distinct fusion phenotypes isolated from infected CCR5−/− patients. Swapping experiments confirmed DR’s low fusion phenotype. Env-mediated fusion is a critical factor among others contributing to long-term survival.

Keywords: HIV fusion, envelope polymorphisms, homozygous, CCR5Δ32

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) utilizes CD4 as a primary receptor and either CCR5 and/or CXCR4 as coreceptors to infect susceptible target cells (reviewed in [1]). R5 viruses primarily use CCR5 as a coreceptor, whereas X4 viruses use CXCR4. R5X4 viruses are dual-tropic and can utilize both coreceptors. R5 strains predominate in early stages during the asymptomatic phase of a subtype B infection, whereas X4 or R5X4 strains can emerge late during the symptomatic phase and are often associated with rapid disease progression.

HIV-1 envelope (Env)-mediated membrane fusion activity is achieved by binding of the Env glycoproteins to CD4 and a coreceptor on the surface of target cells (reviewed in [2]). The Env consists of two non-covalently associated subunits derived by proteolytic cleavage of the gp160 biosynthetic precursor: the gp120 external subunit, which is responsible for binding to specific target cell receptors, and the gp41 transmembrane subunit, which catalyzes the fusion reaction and anchors Env to the viral membrane. Biochemical, genetic, immunochemical, and structural analyses have implicated the sulfated N-terminus and the second extracellular loop of CCR5; on the gp120 side, the bridging sheet and third variable (V3) loop are directly involved. The V3 loop largely determines the coreceptor usage phenotype (R5, X4, R5X4) (reviewed in [2]).

Homozygosity for a 32 bp deletion in CCR5 (CCR5Δ32) is highly associated with protection against HIV-1 infection [3–7]. Although rare, HIV-1 infection of individuals homozygous for the CCR5Δ32 allele (CCR5−/−) has been previously reported [8–17]. All but two of the reported cases showed exclusive use of CXCR4 by virus isolates or the presence of Env sequences typical of CXCR4-using viruses was observed. Despite the lack of CCR5, some of these infected individuals harbored dual tropic R5X4 viruses [10, 11, 18], suggesting that there is no selection against the loss of CCR5 usage.

Previous studies investigating the tropism of the infecting viruses focused on sequence analysis of the gp120 subunit of the HIV-1 Env and in particular the V3 loop region. None of the previous studies examined the sequence of the gp41 subunit of the Envs cloned from these infected CCR5−/− individuals. Infected CCR5−/− subjects harboring X4 viruses are characterized with rapid CD4 T cell loss and disease progression. However, this is not the case when the infecting viruses are dual tropic R5X4 HIV-1 [10, 11, 18]. Churchill et al reported a nef/LTR deleted R5X4 HIV-1 variant in a slow progressor [19, 20]. Previous studies on long-term non-progression (LNTPs) proposed several factors to account for slow disease progression. Unusual polymorphisms were reported in several HIV-1 genes including Gag, Env and Nef in 8 LTNPs [21]. These studies concluded that other viral factors are likely to contribute to modulating the in vivo pathogenicity of HIV-1 strains with nef/LTR deletions.

To determine whether the long-term survival of DR is associated with other factors related to viral entry, the current study analyzed the Env fusion isolated from two infected CCR5−/− patients harboring R5X4 viruses. Envs were cloned from the DR patient 12 years following primary R5X4 infection and the other 1 year after primary infection [10, 11]. A number of unique polymorphisms were identified across the gp41 subunit that were associated with significant differences in Env-mediated fusion activity and delayed onset of syncytia formation. The different fusion phenotypes were confirmed by the transfer of the fusion phenotype upon swapping the sequences encoding the gp41 subunits. The study concludes that the low Env-mediated fusion levels of DR is an important factor among others associated with slow disease progression and/or long-term survival in this CCR5−/− subject.

MATERIAL AND METHODS

Cells and other reagents

The HeLa, 3T3.T4, 3T3.T4.CXCR4, and 3T3.T4.CCR5 cell lines were obtained from the NIH AIDS Reagent and Reference Program. All cell lines were maintained in Dulbecco Modified Eagle Medium (DMEM; Quality Biologicals, Gaithersburg, MD) containing 10% fetal bovine serum (FBS), 2 mM glutamine, and 2 mM penicillin-streptomycin.

Infected Subjects with homozygosity for the CCR5Δ32 allele

Subject DR is a homosexual CCR5−/− male with a history of injecting drug use and first tested seropositive for HIV-1 in September 1991 [11]. The DR HIV-1 Envs were cloned directly from blood taken 12 years after subject DR first tested positive for HIV-1. Subject C2 is another infected CCR5−/− homosexual individual. The clinical history of subject C2 has been previously described [10]. The virus isolate used to clone the C2 Env was obtained from peripheral blood drawn approximately one year after the primary infection. The clinical history of C2 revealed high-risk sexual exposure with two to six male partners during every 6-month interval between visits to the clinic [10].

Sequence Determination and Analysis

The Consensus Clade B was obtained from the LANL HIV Sequence Database (http://www.hiv.lanl.gov/). The complete gp41 regions of the DR and C2 clones were sequenced using the capillary sequencing at Functional Biosciences Inc, Madison, WI. The complete gp41 sequences were constructed using the CAP3 webpage [22] and manual trimming. The sequences were then translated into predicted amino acids, aligned using Clustal W [23], and then displayed by EMBOSS show align [24]. Determination of the HR2 region’s net charge was done by using the ExPASy server’s ProtParam [25]. A BLAST search of the HIV Sequence Database was performed with the HR2 region and flanking sections to identify homologous sequences.

Construction of hybrid C2 and DR8 Envs

The C2 and DR8 hybrid Envs were constructed by exchanging most of the gp41 region. C2–22 and DR Env clones were digested with BbvC1 enzyme and swapped the 810 bp fragment, which includes both gp41 HR1 and HR2 regions. The resulting recombinant Envs containing the swapped domains were sequenced and confirmed for the presence of the swapped gp41 region. The V3 swapped mutants were constructed by Mutagenix (Grand Island, NY). The resulting recombinant Envs containing the swapped domain were sequenced and confirmed for the presence of the swapped V3 domain.

Env-mediated fusion and detection of syncytia formation

All Env-mediated fusion assays described in this study were conducted as previously described [26, 27]. All Envs were cloned under early/late vaccinia promoter. Effector Hela cells were transfected with 5ug Env-expressing plasmid. Env expression was activated by infecting transfected cells with PT7-lacZ vaccinia virus (lacZ reporter gene under T7 promoter). The target NIH 3T3-CD4, 3T3-CD4-CXCR4, 3T3-CD4-CCR5 cells were infected by vaccinia virus encoding the bacteriophage T7 RNA polymerase. The pSC-59 plasmid encoding an uncleaved Env was used to measure the non-specific background in the fusion assay. After one hour of infection at 37 0C and incubation at 310C for 16 hours, the Env-expressing Hela cells were mixed with the target cells at 1:1 ratio. The cell mixtures were incubated for the indicated times (60 min, 90min or 150 min) at 37 0C and syncytia monitored using an EVOS core XL phase contrast microscope at the same time another portion of the same fusion mix was lysed by 10% NP-40 and the substrate chlorophenolred-b-D-galactopyranoside (CPRG) was added. The VersaMax microplate spectrophotometer (Molecular Devices) was used to measure the extent of Env-mediated fusion over time with multiple readings taken at regular intervals at a wavelength of 590nm. Values are reported as optical density (ODX1000/min).

T20 was added at increasing concentrations to the Env-expressing cells before they were mixed with 3T3.T4.CCR5 or 3T3.T4.CXCR4 target cells. The blocking activity of T20 was analyzed by measuring the extent of β-galactosidase produced at each T20 concentration.

Single-round infection assays

HIV-1 pseudotyped viral stocks were prepared as previously described [28]. Producer 293T cells were transfected with HIV-Luc+Env− and another plasmid DNA containing the desired HIV-1 Env under CMV promoter. Pseudotyped viral particles were harvested from the culture supernatants after 60–72 hr and tittered by measuring the amount of p24 gag viral antigen. Pseudotyped virus infection of either 3T3.T4.CCR5 or 3T3.T4.CXCR4 was performed in 48-well culture dishes (5×104 cells/well) at 50ng of P24-normalized virus. The infections were incubated for 36–40 hr at 37ᵒC with 5% CO2. Infectivity was evaluated by measuring luciferase production.

FACS analysis and western blot analysis

This study used T50, a monoclonal antibody (Mab) to gp120 (provided by Chris Broder) to analyze surface expression of HeLa cells expressing the different HIV-1 Envs. Env-expressing cells were reacted with the gp120 Mab, washed, then reacted with PE-conjugated anti-mouse IgG, washed and analyzed by FACS Calibur.

Total cell lysates of DR or C2-expressing cells were used for the western blot analysis. Cells were resuspended in protein lysis buffer (300mM NaCL, 50mM Tris.HCL pH 7.6, 0.1% SDS, Aprotonin 10ug/ml, PMSF 1mM) and fractionated in a 8% SDS-PAGE (~0.1×106cells/lane) and transferred into PVDF membrane blots. The blots were reacted with goat anti-gp120 polyclonal antibodies (AIDS Reagent Program, Germantown, MD). Env proteins were visualized using horseradish peroxidase-conjugated anti-goat immunoglobulin G antibodies (Santa Cruz Biotech., CA) and SuperSignal enhanced chemiluminescence (GE Healthcare).

RESULTS

Isolation, sequencing, and expression of cloned DR and C2 Envs

Sequencing of the gp41 regions of the eight Env clones demonstrated significant polymorphisms in comparison to the consensus gp41 sequence. Significant Polymorphisms were observed in the HR1, HR2 domain, however, scattered polymorphisms were also observed in other gp41 regions (Fig. 1). The gp41 region had an insertion of the amino acids STNIP found before the HR2 region in the DR Env clones (Fig. 1). The Clade B consensus sequence and the C2 clones have a net charge of −7 based on the simple addition of positive and negative charges of the amino acids within the HR2 region. The DR1, DR8 and DR17 HR2 regions have a lower net negative charge of −3, while DR19 has a net charge of −2. This difference in the net negative charge of the HR2 region is due to an increase in the number of positively charged amino acids along with a decrease in the number of negatively charged amino acids in the HR regions of DR clones. Therefore, the net charge of the HR2 domains of the C2 Envs is significantly higher than the corresponding HR2 domains of the DR Envs. It is also noted that all DR clones were missing a methionine residue in the fusion peptide (FP) and had a proline instead of the glycine found in all C2 Env clones (Fig. 1, FP region).

Figure 1.

Figure 1.

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Sequence alignments of the gp41 regions of C2 and DR Env clones with the consensus Clade B sequence from the HIV Database. The key to the alignment is as follows: ‘*’ represent exact matches with the consensus B sequence, ‘-’ are gaps in the sequence, and letters are differences within the sequence when compared to the consensus clade B sequence. Sequence alignments of gp41 subunit using the Clade B consensus sequence as a reference.

DR Envs show significantly lower fusion levels in cell fusion and pseudotyped virus infection assays

An experimental approach that involves the analysis of fusion between two distinct cell populations was used to compare Env-mediated fusion levels; one expressing CD4 (endogenous or vaccinia virus encoded) and the other expressing the indicated HIV-1 Env (vaccinia virus encoded). Fusion was scored by a reporter gene activation assay in which the cytoplasm of one cell population contained vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other contained the lacZ gene linked to the T7 promoter; cell fusion activates β-Gal [29]. In comparison to DR Envs, the C2 Env clones consistently produced a significantly higher signal in the Env-mediated fusion assay with target cells expressing either CCR5 or CXCR4 (Fig. 2A).

Figure 2.

Figure 2.

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Env-mediated fusion and pseudotyped virus infection. A) HeLa cells were transfected with the indicated Envs and infected with vCB-21R (encoding β-gal under T7 promoter). The target cells (3T3.T4.CXCR4 or 3T3.T4.CCR5) were infected with vTF7–3 (encoding T7 RNA polymerase). Both cells were mixed and incubated for 2.5 hours, then measured by quantifying the β-galactosidase produced. The results are representative of at least ten different experiments performed in duplicates. B) FACS analysis of cell surface Env in transfected HeLa cells used in the fusion assay shown in A. Cells were reacted with the T50 monoclonal antibody and stained with FITC-conjugated secondary antibody. The cell surface staining results are represented as mean fluorescence intensity. C) Western blot analysis Env expressing either C2 or DR Envs used in A. D) The indicated Envs were used to generate pseudotyped viruses used to infect either 3T3.T4.CR5 or 3T3.T4.CXCR4. The results, expressed as relative light units (RLU) show a representative of three different experiments performed in triplicates. Values in A &D were expressed as mean ± standard deviation. p-values were calculated using Student’s T-test.

FACS analysis of DR and C2 transfected HeLa cells gated on live cells demonstrated comparable cell surface protein expression levels of all Env proteins (Fig.2B). Western blot analysis was performed to confirm the proper cleavage of Envs (Fig. 2C). The results show that the higher Env-mediated fusion activity of the C2 Envs is not due to either cleavage efficiency or Env expression levels.

A single-round infection assay was used to determine the infectivity of the DR and C2 pseudotyped viruses. DR infectivity was significantly lower than reference 89.6 or C2 Envs as measured by luciferase activity (Fig. 2D). The DR Envs showed less potency in both Env-mediated fusion and pseudotyped virus infection, two different assays that model the entry stage of the HIV-1 life cycle.

The DR Envs show delayed onset and smaller size syncytia

Syncytia formation was monitored over time to determine whether the high levels of Env-mediated fusion by C2 Envs is due to earlier onset of syncytia formation. A syncytia formation assay was performed to microscopically monitor syncytia formation over time following mixing Env-expressing cells with the target cells. No syncytia formation was observed with the DR Envs at 60 min (Fig. 1A Suppl.). The DR Envs developed small size syncytia at 90 min (Fig.1B Suppl. and 150 min (Fig. 1C Suppl.) post cell mixing. In contrast, syncytia formation was readily observed 60 min with cells expressing the C2 Envs (Fig. 1D Suppl.) Earlier appearance and larger size syncytia were consistently observed with the C2 Envs at 90 min (Fig. 1E Suppl.) and 150 min (Fig. 1F, Suppl.) following cell mixing. Similar results were obtained when 3T3.T4.CXCR4 cells were used as the target cells (data not shown). These results suggested that the higher fusion mediated by the C2 Envs could be due to the earlier onset and larger size of syncytia. The β-galactosidase activity of the same fusion mixtures was measured at the same time the photos were taken to confirm the cell fusion observed in the photomicrographs. The fusion activity levels correlated with the observed number and size of syncytia (Fig. 1G-I, Suppl.).

The DR and C2 Envs show different sensitivities to the fusion inhibitor T20

T20 blocking experiments were performed to determine whether the polymorphic gp41 sequences impact the Inhibition concentration 50 (IC50) of Env-mediated cell fusion. This assay is dependent on measuring the T20 blocking activity at different time points following mixing the Env-expressing and target cells. FACS analysis confirmed comparable cell surface expression of C2 and DR Env proteins in transfected/infected cells.

T20 treatments determined that the IC50 of the C2 Envs ranged between 0.05–0.1 μg/ml (Fig. 3A&C). The IC50 for the DR Envs was at least 10 times higher and ranged from 1.0–1.1 μg/ml (Fig 3B&D). The results indicate a significant difference between the C2 and DR Envs in terms of their sensitivity to the fusion peptide mimic T20.

Figure 3.

Figure 3.

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T20 inhibition of C2 and DR Env-mediated fusion. The indicated Env-expressing cells were prepared as mentioned in Figure 2. Expression of Envs was confirmed by FACS analysis as described in methods. T20 was added at the indicated concentrations to Env-expressing cells before mixing with the target 3T3.T4.CCR5 cells (A&B) or 3T3.T4.CXCR4 cells (C&D). The mixtures were incubated for 2.5 hr and the inhibitory effect of T20 was assessed by measuring β-galactosidase activity. The results are representative of at least five different experiments performed in triplicates. Relative fusion values are shown. Results without T20 treatment were considered as 100% fusion. The absolute fusion values for Envs without T20 treatment are: For CCR5 cells: C2–16, 280; C2–22, 265; DR-8, 61; DR-1, 51. For CXCR4 cells: C2–16, 154; C2–22, 131; DR-8, 45; DR-1, 38.

Swapping the gp41 subunits but not the V3 domain results in the transfer of the Env-mediated fusion phenotype

To determine whether the gp41 polymorphisms had an impact on the Env fusion activity, we next tested whether swapping the polymorphic g41 subunit or V3 domain would result in gain or loss of Env-mediated fusion activity (Fig. 4A). The fusion results demonstrated that the hybrid Envs with the swapped gp41 subunit showed the transfer of the fusion phenotype. For example, compared to the parental DR Env, the DR Env containing the C2–22 gp41 region showed higher Env-mediated fusion activity (Fig. 4B). In contrast, compared to the parental C2–22 Env, the C2–22 Env containing the DR gp41 region showed lower fusion activity (Fig. 4B). In contrast, swapping the V3 domains did not result in a significant change in the Env fusion phenotype (Fig. 4B). FACS analysis of Env-expressing cells indicated comparable Env surface expression (Fig 4B, MFI).

Figure 4.

Figure 4.

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Swapping the V3 or the gp41 domains. A) Schematic diagram showing the hybrid Envs with swapped domains. The numbers indicate the position of the amino acids according to the predicted primary amino acid sequences of C2 and DR Envs. B) Env-mediated fusion activity of the hybrid Envs. Env-expressing cells were mixed with the indicated targets (either 3T3.T4.CXCR4 or 3T3.T4.CCR5) and incubated for 2.5 hr. The Env-mediated fusion activity was analyzed by measuring β-galactosidase produced in fused cells. The numbers below each Env represents the mean fluorescence intensity of each Env at the time of mixing Env-expressing cells with the targets. The results are representative of at least three different experiments performed in triplicates.

T20 was added at increasing doses to Env-expressing cells prior to cell mixing to confirm the transfer of the fusion phenotype by the swapped gp41 Envs. The results indicated that the IC50 of the swapped Envs correlated with the source of the gp41 region (Fig. 5). For example, the IC50 of C2 Env containing the DR gp41 (C2-DR8gp41) was at least 10 times higher than the IC50 of the parental C2–22 Env (Fig. 5 A&C). In contrast, the IC50 of the DR Env containing the C2–22 gp41 region was lower than that of the parental DR8 Env (Fig. 5 B&D). The results indicated that the sensitivity to T20 was transferred by swapping the gp41 regions provided further evidence for the distinct fusion phenotypes of C2 and DR Envs and confirmed DR’s low Env fusion phenotype.

Figure 5.

Figure 5.

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T20 inhibition of Env-mediated fusion by swapped Envs. The indicated Env-expressing cells were prepared as described earlier. Expression of Envs was confirmed by FACS analysis. T20 was added at the indicated concentrations to Env-expressing cells before mixing with the target 3T3.T4.CCR5 cells (A&B) or 3T3.T4.CXCR4 cells (C&D). The mixtures were incubated for 2.5 hr and the inhibitory effect of T20 was assessed by measuring β-galactosidase produced in fused cells. The results are representative of at least three different experiments performed in triplicates. Relative fusion values are shown. Results without T20 treatment were considered as 100% fusion. The absolute fusion values without T20 treatment are: CCR5 cells: C2–22, 205; C2-DR8gp41, 68; DR8, 58; DR8-C2gp41, 135; CXCR4 cells: C2–22, 142; C2-DR8gp41, 53; DR8, 42; DR8-C2gp41, 83.

DISCUSSION

This study reports novel gp41 subunit sequences derived from eight R5X4 Env glycoproteins cloned from two CCR5−/− individuals (C2 and DR). We demonstrated that the gp41 polymorphisms of two infected CCR5−/− patients translated into two distinct fusion phenotypes. The observed gp41 polymorphisms were not associated with resistance to fusion inhibitors because the patients were naïve for enfuvirtide therapy [11]. The low Env-mediated fusion phenotype is associated with DR Envs, derived from a chronically infected subject who is a long-term survivor. The observed significant differences in the Env-mediated fusion activities were not due to differences in Env expression or processing because all Envs were comparably expressed at the cell surface demonstrated by FACS analysis using monoclonal antibodies to gp120. The Env-mediated fusion results reported in this study are different from those previously published by Gray et al [11]. This is due to differences in the experimental make-up, including plasmid vector backbones, target cell lines, Target:Env ratio, and incubation periods of the Env fusion mix. For example, Gray et al incubated the Env fusion mix overnight, while the current study incubated the fusion mix for a maximum of 2.5 hours. In addition, they used a 10:1 target:Env ratio while our assay used 1:1 ratio. In pseudotyped virus infections, they used cf2 cells and measured luciferase at 72 hr postinfection while this study used 3T3.T4 cells and measured luciferase at 36–40 hr postinfection.

A BLAST search of the STNIP and the gp41 region of the DR Env clones returned three hits (87–96% identical) of three progressors: HIV Sequence Database loci AF042105.1, AF042102.1, and AF042106.1. Like DR these also had a net charge of −3, and another hit (92% identical) of HIV-1 sequences from four members of the Sydney Blood Bank Cohort of long term non-progressors (AAD03239.1). The BLAST search also returned another two hits (91% identical) of two isolates, one from an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients [30], and another from an impaired fitness study of acute/early viruses in persons who became HIV controllers. These hits were not returned when the BLAST search was performed using the C2–22 gp41 sequence. In contrast, a BLAST search of the gp41 region of the C2 Env clones returned different hits (93% identical) of HIV-1 Envs that are consistent with more pathogenic viruses, HIV Sequence Database loci U69593.1 [31], L02317.1 [32], and HQ699978.1 [33]. Similar to the DR and C2 polymorphisms concentrated in the HR2 region, previous studies reported unusual polymorphisms in the gp41 regions of 8 LTNPs with a significant number occurring in the HR2 region [21].

The V3 loop of HIV-1 gp120 has been intensively studied to analyze interactions with the coreceptors and predicting coreceptor usage of HIV-1 isolates [34–37]. Since both DR and C2 Envs are R5X4 swapping the V3 loop did not result in a significant change in Env fusion. We used the V3 swapping as a control to confirm the distinct Env fusion phenotypes of DR and C2. As expected the V3 loop swap did not significantly change the T20 IC50 of Env fusion. However, swapping the gp41 regions of DR and C2 resulted in the transfer of the T20 IC50 indi4cating two distinct fusion phenotypes. Previous studies have demonstrated the importance of the HR domain in Env fusion kinetics. Sivaraman et al demonstrated that the heptad repeat 2 domains is the major determinant for enhanced HIV-1 fusion and pathogenicity [38]. Other studies demonstrated that enfuvirtide-resistant HR2 polymorphism N140I combined with the HR1 V38A mutation is associated with a less cytopathic phenotype [39]. It is possible that the higher net negative charge of the HR2 domain of the C2 Envs is associated with these observed functional differences. The higher negative charge may promote better interaction of the HR2 domain with the host cell membrane, leading to higher Env-mediated fusion signals.

Previous studies by Shioda et al have suggested the association of V2 extensions with slow disease progression [40]. Other studies by Wang et al indicated that HIV-1 strains from a cohort of American subjects showed a V2 region extension unique to slow progressors and long term non-progressors (LTNP) [41]. The DR V2 extension introduced 4 new N-linked glycosylation sites in the gp120 subunit [11]. It has previously been demonstrated that V1-V2 loop sequences expand and add glycosylation sites over the course of infection, and that these modifications affect antibody neutralization sensitivity [42]. Sagar et al suggested that changes within the V1-V2 domains over the course of infection might impact host/receptor/coreceptor interaction [42]. Hybrid Envs containing swapped V2 domains might provide an insight into the role of the V2 extension in Env-mediated fusion activity.

It is also possible that viral adaptation to grow in CCR5−/− cells resulted in polymorphisms that favor slow disease progression in some of the patients harboring R5X4 viruses. The in vivo importance of CCR5 density in determining viral load has previously been established. Reynes et al. postulated that CCR5 expression affects virus production and viral load [43], and found a strong correlation between CCR5 density and viral load. Platt et al. elucidated a threshold of 10,000 CCR5 molecules per CD4+ cell as a requirement for HIV infection [44]. Most individuals with a low viral load have CCR5 densities below this value [43]. The persistence of R5 viruses in a CCR5-deficient subject suggests that structural features of Env associated with CCR5 usage may allow HIV-1 persistence through a non CCR5-dependent mechanism. It will be interesting to investigate whether similar fusion phenotypes can be isolated from CCR5+/+ patients.

Supplementary Material

Supplemental Data File _.doc_.tif_pdf_etc._

Supplementary Figure 1. Time course of syncytia formation. The indicated Env plasmids were transfected into HeLa cells and Env expression activated by infection with vCB-21R. The 3T3.T4.CXCR4 or 3T3.T4.CCR5 cells were infected with vTF7–3 and used as targets. Env-expressing cells were mixed with the target cells. Syncytia were analyzed at 60 min (A&D), 90 min (B&E) and 150 min (C&F) of incubation at 37?C. Syncytia were microscopically monitored and photographed by using an EVOS core XL phase contrast microscope. Arrows point to cells involved in syncytia. A representative field is shown for each Env. Panels G-I show the results of Env-mediated fusion of the same Env-target mixtures analyzed by measuring ?-galactosidase produced at the indicated time points post-mixing.

Acknowledgments

We thank Dr. Chris Broder for providing T50 and other monoclonal antibodies to the gp120 protein. Special thanks go to Andy Grass, Viswanathan Rajagopalan, Troy Camarata, and Carol Brenner for excellent comments. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: T20, Fusion Inhibitor from AIDS, NIAID; HIV-1 gp160 Antiserum (HT7); 3T3.T4, 3T3.T4.CXCR4, and 3T3.T4.CCR5 from Dr. Dan Littman. This work was supported by a NIH grant # AI052019 awarded to GA.

The C2 and DR Env nucleotide sequences reported here have been assigned GenBank accession numbers KM061046 (C2–3), KM061047 (C2–16), KM061048 (C2–22), KM061049 (C2–24), KM061050 (DR-1), KM061051 (DR-8), KM061052 (DR-17), KM061053 (DR-19).

Funding: This work was supported by a NIH grant # AI052019 awarded to GA.

Footnotes

Potential conflict of interest: The authors declare they have no conflict of interest to disclose.

References

  • 1.Alkhatib G Counterpoint: cord blood stem cell therapy for acquired immune deficiency syndrome. Stem Cells Dev 2009; 18(1):5–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Alkhatib G The biology of CCR5 and CXCR4. Curr Opin HIV AIDS 2009; 4(2):96–103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 1996; 273(5283):1856–1862. [DOI] [PubMed] [Google Scholar]
  • 4.Huang Y, Paxton WA, Wolinsky SM, Neumann AU, Zhang L, He T, et al. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med 1996; 2(11):1240–1243. [DOI] [PubMed] [Google Scholar]
  • 5.Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996; 86(3):367–377. [DOI] [PubMed] [Google Scholar]
  • 6.Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene [see comments]. Nature 1996; 382(6593):722–725. [DOI] [PubMed] [Google Scholar]
  • 7.Zimmerman PA, Buckler-White A, Alkhatib G, Spalding T, Kubofcik J, Combadiere C, et al. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk. Molecular Medicine 1997; 3(1):23–36. [PMC free article] [PubMed] [Google Scholar]
  • 8.Balotta C, Bagnarelli P, Violin M, Ridolfo AL, Zhou D, Berlusconi A, et al. Homozygous delta 32 deletion of the CCR-5 chemokine receptor gene in an HIV-1-infected patient. Aids 1997; 11(10):F67–71. [DOI] [PubMed] [Google Scholar]
  • 9.Biti R, Ffrench R, Young J, Bennetts B, Stewart G, Liang T. HIV-1 infection in an individual homozygous for the CCR5 deletion allele [letter; comment]. Nature Medicine 1997; 3(3):252–253. [DOI] [PubMed] [Google Scholar]
  • 10.Gorry PR, Zhang C, Wu S, Kunstman K, Trachtenberg E, Phair J, et al. Persistence of dual-tropic HIV-1 in an individual homozygous for the CCR5 Delta 32 allele. Lancet 2002; 359(9320):1832–1834. [DOI] [PubMed] [Google Scholar]
  • 11.Gray L, Churchill MJ, Keane N, Sterjovski J, Ellett AM, Purcell DF, et al. Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele. J Virol 2006; 80(7):3684–3691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Heiken H, Becker S, Bastisch I, Schmidt RE. HIV-1 infection in a heterosexual man homozygous for CCR-5 delta32. AIDS 1999; 13(4):529–530. [DOI] [PubMed] [Google Scholar]
  • 13.Iversen AK, Christiansen CB, Attermann J, Eugen-Olsen J, Schulman S, Berntorp E, et al. Limited protective effect of the CCR5Delta32/CCR5Delta32 genotype on human immunodeficiency virus infection incidence in a cohort of patients with hemophilia and selection for genotypic X4 virus. J Infect Dis 2003; 187(2):215–225. [DOI] [PubMed] [Google Scholar]
  • 14.Kuipers H, Workman C, Dyer W, Geczy A, Sullivan J, Oelrichs R. An HIV-1-infected individual homozygous for the CCR-5 delta32 allele and the SDF-1 3’A allele. Aids 1999; 13(3):433–434. [DOI] [PubMed] [Google Scholar]
  • 15.O’Brien TR, Winkler C, Dean M, Nelson JA, Carrington M, Michael NL, et al. HIV-1 infection in a man homozygous for CCR5 delta 32 [letter] [see comments]. Lancet 1997; 349(9060):1219. [DOI] [PubMed] [Google Scholar]
  • 16.Sheppard HW, Celum C, Michael NL, O’Brien S, Dean M, Carrington M, et al. HIV-1 infection in individuals with the CCR5-Delta32/Delta32 genotype: acquisition of syncytium-inducing virus at seroconversion. J Acquir Immune Defic Syndr 2002; 29(3):307–313. [DOI] [PubMed] [Google Scholar]
  • 17.Theodorou I, Meyer L, Magierowska M, Katlama C, Rouzioux C. HIV-1 infection in an individual homozygous for CCR5 delta 32. Seroco Study Group [letter] [see comments]. Lancet 1997; 349(9060):1219–1220. [PubMed] [Google Scholar]
  • 18.Naif HM, Cunningham AL, Alali M, Li S, Nasr N, Buhler MM, et al. A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR5Delta32 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4. J Virol 2002; 76(7):3114–3124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Churchill M, Sterjovski J, Gray L, Cowley D, Chatfield C, Learmont J, et al. Longitudinal analysis of nef/long terminal repeat-deleted HIV-1 in blood and cerebrospinal fluid of a long-term survivor who developed HIV-associated dementia. J Infect Dis 2004; 190(12):2181–2186. [DOI] [PubMed] [Google Scholar]
  • 20.Churchill MJ, Rhodes DI, Learmont JC, Sullivan JS, Wesselingh SL, Cooke IR, et al. Longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source. J Virol 2006; 80(2):1047–1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Alexander L, Weiskopf E, Greenough TC, Gaddis NC, Auerbach MR, Malim MH, et al. Unusual polymorphisms in human immunodeficiency virus type 1 associated with nonprogressive infection. J Virol 2000; 74(9):4361–4376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kostrikis LG, Neumann AU, Thomson B, Korber BT, McHardy P, Karanicolas R, et al. A polymorphism in the regulatory region of the CC-chemokine receptor 5 gene influences perinatal transmission of human immunodeficiency virus type 1 to African-American infants. Journal of Virology 1999; 73(12):10264–10271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007; 23(21):2947–2948. [DOI] [PubMed] [Google Scholar]
  • 24.Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 2000; 16(6):276–277. [DOI] [PubMed] [Google Scholar]
  • 25.Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, et al. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 1999; 112:531–552. [DOI] [PubMed] [Google Scholar]
  • 26.Jin Q, Agrawal L, Meyer L, Tubiana R, Theodorou I, Alkhatib G. CCR5Δ32 59537-G/A promoter polymorphism is associated with low translational efficiency and the loss of CCR5Δ32 protective effects. J Virol 2008; 82(5):2418–2426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Jin Q, Altenburg JD, Hossain MM, Alkhatib G. Role for the conserved N-terminal cysteines in the anti-chemokine activities by the chemokine-like protein MC148R1 encoded by Molluscum contagiosum virus. Virology 2011; 417(2):449–456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Jin Q, Agrawal L, VanHorn-Ali Z, Alkhatib G. Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: evidence using antibodies specific to the receptor’s large extracellular domain. Virology 2006; 349(1):184–196. [DOI] [PubMed] [Google Scholar]
  • 29.Nussbaum O, Broder CC, Berger EA. Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation. JVirol 1994; 68:5411–5422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, et al. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 1995; 270(5238):988–991. [DOI] [PubMed] [Google Scholar]
  • 31.Fang G, Burger H, Chappey C, Rowland-Jones S, Visosky A, Chen CH, et al. Analysis of transition from long-term nonprogressive to progressive infection identifies sequences that may attenuate HIV type 1. AIDS Res Hum Retroviruses 2001; 17(15):1395–1404. [DOI] [PubMed] [Google Scholar]
  • 32.Ghosh SK, Fultz PN, Keddie E, Saag MS, Sharp PM, Hahn BH, et al. A molecular clone of HIV-1 tropic and cytopathic for human and chimpanzee lymphocytes. Virology 1993; 194(2):858–864. [DOI] [PubMed] [Google Scholar]
  • 33.Shang H, Han X, Shi X, Zuo T, Goldin M, Chen D, et al. Genetic and neutralization sensitivity of diverse HIV-1 env clones from chronically infected patients in China. J Biol Chem 2011; 286(16):14531–14541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Chesebro B, Wehrly K, Nishio J, Perryman S. Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes. J Virol 1996; 70(12):9055–9059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Hoffman NG, Seillier-Moiseiwitsch F, Ahn J, Walker JM, Swanstrom R. Variability in the human immunodeficiency virus type 1 gp120 Env protein linked to phenotype-associated changes in the V3 loop. J Virol 2002; 76(8):3852–3864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Shioda T, Levy JA, Cheng-Mayer C. Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 1992; 89(20):9434–9438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Willey RL, Theodore TS, Martin MA. Amino acid substitutions in the human immunodeficiency virus type 1 gp120 V3 loop that change viral tropism also alter physical and functional properties of the virion envelope. J Virol 1994; 68(7):4409–4419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Sivaraman V, Zhang L, Meissner EG, Jeffrey JL, Su L. The heptad repeat 2 domain is a major determinant for enhanced human immunodeficiency virus type 1 (HIV-1) fusion and pathogenicity of a highly pathogenic HIV-1 Env. J Virol 2009; 83(22):11715–11725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Cunyat F, Marfil S, Garcia E, Svicher V, Perez-Alvarez N, Curriu M, et al. The HR2 polymorphism N140I in the HIV-1 gp41 combined with the HR1 V38A mutation is associated with a less cytopathic phenotype. Retrovirology 2012; 9:15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Shioda T, Oka S, Xin X, Liu H, Harukuni R, Kurotani A, et al. In vivo sequence variability of human immunodeficiency virus type 1 envelope gp120: association of V2 extension with slow disease progression. J Virol 1997; 71(7):4871–4881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wang B, Spira TJ, Owen S, Lal RB, Saksena NK. HIV-1 strains from a cohort of American subjects reveal the presence of a V2 region extension unique to slow progressors and non-progressors. AIDS 2000; 14(3):213–223. [DOI] [PubMed] [Google Scholar]
  • 42.Sagar M, Wu X, Lee S, Overbaugh J. Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity. J Virol 2006; 80(19):9586–9598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Reynes J, Portales P, Segondy M, Baillat V, André P, Réant B, et al. CD4+ T Cell Surface CCR5 Density as a Determining Factor of Virus Load in Persons Infected with Human Immunodeficiency Virus Type 1. The Journal of Infectious Diseases 2000; 181(March). [DOI] [PubMed] [Google Scholar]
  • 44.Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. Journal of Virology 1998; 72(4):2855–2864. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Data File _.doc_.tif_pdf_etc._

Supplementary Figure 1. Time course of syncytia formation. The indicated Env plasmids were transfected into HeLa cells and Env expression activated by infection with vCB-21R. The 3T3.T4.CXCR4 or 3T3.T4.CCR5 cells were infected with vTF7–3 and used as targets. Env-expressing cells were mixed with the target cells. Syncytia were analyzed at 60 min (A&D), 90 min (B&E) and 150 min (C&F) of incubation at 37?C. Syncytia were microscopically monitored and photographed by using an EVOS core XL phase contrast microscope. Arrows point to cells involved in syncytia. A representative field is shown for each Env. Panels G-I show the results of Env-mediated fusion of the same Env-target mixtures analyzed by measuring ?-galactosidase produced at the indicated time points post-mixing.

NIHMS980346-supplement-Supplemental_Data_File___doc___tif__pdf__etc__.pptx (938.3KB, pptx)

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