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Published as doi: 10.1096/fj.07-9576com.
 (The FASEB Journal. 2008;22:1179-1192.)
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(The FASEB Journal. 2008;22:1179-1192.)
© 2008 FASEB

The function of coreceptor as a basis for the kinetic dissection of HIV type 1 envelope protein-mediated cell fusion

Miao-Ping Chien*, Shibo Jiang{dagger} and Ding-Kwo Chang*,1

* Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China; and

{dagger} Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA

1Correspondence: Institute of Chemistry, Academia Sinica, Taipei, Taiwan 11529, Republic of China. E-mail: dkc{at}chem.sinica.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event.—Chien, M.-P., Jiang, S., Chang, D.-K. The function of coreceptor as a basis for the kinetic dissection of HIV type 1 Env-mediated cell fusion.


Key Words: gp120 shedding • NC-1 sensitive conformation • lipid and content mixing • recruitment • fusion peptide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV-1 GAINS ENTRY INTO SUSCEPTIBLE cells by means of fusion of viral membrane with the plasma membrane (1) , which is mediated by the envelope glycoprotein (gp). HIV-1 envelope (Env) protein is a homotrimer of a heterodimer consisting of surface gp120 and transmembrane gp41 subunits (2) . The fusion reaction is driven by interaction of gp120/gp41 with the host cell surface CD4 (3) , which induces conformational changes in gp120, mainly in the V3 loop and the bridging sheets, to facilitate binding with the coreceptor (4 , 5) . HIV-1 primarily uses CCR5 (R5) or CXCR4 (X4) (6 7 8 9) as the coreceptor that is the determinant of viral tropism and implicated in viral pathogenesis. Formation of the ternary receptor-coreceptor-Env complex further triggers a cascade of conformational changes in the Env glycoprotein (10 , 11) , exposing the hydrophobic fusion peptide (FP) region and the formation of a trimeric helix hairpin in gp41, eventually leading to fusion. Immunological studies also implicated mutations in the receptor and coreceptor binding sites within gp120 in the neutralization-resistance phenotype (12) . As such the coreceptor is pivotal to the virus-mediated membrane fusion and its interaction with Env a potential therapeutic target.

However, the function of the coreceptor at the molecular level remains incompletely understood. For instance, the CD4-induced conformational changes in gp41 entail the exposure of the prehairpin intermediate, but how does the coreceptor induce additional conformational changes in gp120/gp41? Specifically, is gp120 shedding promoted by the coreceptor engagement and, if so, does it precede prehairpin formation of gp41? Also, when does the exposure of the N-terminal FP of gp41 occur temporally relative to the exposure of the prehairpin structure or the shedding of gp120? Which of the fusogenic steps is dependent on the coreceptor? Understanding these issues could shed light on the fusion mechanism and aid anti-HIV-1 therapeutics. Some studies have addressed these questions by indirectly probing the effect of conformational changes on the reactivity of antibodies or inhibitors, as manifested in cell fusion (10 , 13 , 14) . However, the cell fusion assay was unable to monitor in real time the kinetics of antibodies or inhibitors binding to the epitopes accessible on receptor-coreceptor engagement.

In the present work, we have developed an approach to examine the conformational changes in Env, including gp41 N- and C-helix exposure (prehairpin) and gp120 shedding by using the optical biosensor, surface plasmon resonance (SPR), a class of analytical instruments that detect interactions between molecules and changes at the sensor surface in real time. We used the L1 sensor chip, which consists of a carboxymethyldextran hydrogel with lipophilic alkyl chain anchors on the gold surface. Liposomes containing detergent-resistant rafts (raft-liposomes) (15 16 17) were passed over and captured by the hydrophobic anchors. HIV-1 gp120/gp41 incubated with raft-liposomes was immobilized on the L1 sensor chip, providing a chemically and physically stable environment to mimic the Env expressed on the viral surface, allowing immediate monitoring of the Env conformational change by sensorgrams instead of indirectly by the reaction with antibodies or inhibitors. Moreover, to monitor the various steps leading to fusion, we also used live cell imaging fluorescence to observe the kinetics of the exposure and insertion of FP, the recruitment of Env, and lipid and content mixing. The technique can analyze events real time at the effector (Env-expressing HeLa) and target (CD4 or CD4-X4 NIH3T3) cells. In addition, kinetics of exposure and membrane insertion of the fusion peptide domain of gp41 was detected using medium polarity-sensitive 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence.

Remarkably, it was found that shedding of gp120 requires X4, which is also needed for lipid and aqueous content mixing. On the other hand, exposure and membrane insertion of FP and the prehairpin structure can proceed with CD4 engagement only. The ectodomain bundle formation as probed by antibodies NC-1 was accelerated by X4. The real-time monitoring of these key steps with fluorescence imaging microscopy allows kinetic determination of the temporal order thereof and construction of a refined model of Env-mediated fusion, which may serve as a paradigm of other class I fusion proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, plasmids, antibodies, and inhibitors
The 293T (from a human embryonic kidney cell line) cells and HXB2 gp160-pSVE7, CD4-pcDNA3, and pEGFP-N1 plasmids and their antibodies were gifts from S. S. Chen (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China). The HeLa cells, NIH3T3 cells, X4 plasmids (fusin-pcDNA1), and X4 antibodies were obtained through the U.S. National Institutes of Health AIDS Research and Reference Reagent Program. The cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (GIBCO Life Technologies, Inc., Grand Island, NY, USA), penicillin and streptomycin.

Expression and purification of gp120/gp41, CD4, and X4
HXB2 gp120/gp41, CD4, and X4 proteins were prepared from 293T cells (5x106), which were transiently transfected using Lipofectamine (Invitrogen, Carlsbad, CA, USA) by HXB2 gp160-pSVE7 plasmids, CD4-pcDNA3, and X4-pcDNA1, respectively. After 48 h of expression, cells were washed twice with cold (4°C) PBS, pelleted by centrifugation, and resuspended in lysis buffer (5 mM iodoacetamide, 150 mM NaCl, 20 mM Tris, 20 mM EDTA, 1% Brij97, and protease inhibitors) at 4°C for 40 min with gentle mixing. The nuclei were then pelleted by centrifugation at 20,000 g for 20 min in a refrigerated centrifuge. Protein G-Sepharose beads (Sigma-Aldrich Corp., St. Louis, MO, USA) coated with each of the aforementioned antibodies before incubation with cell lysate were prewashed with PBS and added to the cell lysate at 4°C for 14 h. The beads were then washed five times with lysis buffer to remove the unbound proteins. The proteins were subsequently eluted by 100 mM glycine-HCl (pH 2.8) and immediately neutralized with 1 M Tris base. The expression proteins were checked by Western blot analysis.

Measurements of kinetics of FP exposure and insertion
Bis-ANS (Sigma-Aldrich Corp.) (excitation, 395 nm; emission, 500 nm) was added as the probe of FP exposure to HIV-1 Env-expressing cells at 4 µM in medium without serum in an open cell chamber. CD4-X4- or CD4-alone-expressing NIH3T3 (target) cells were added after steady levels of fluorescence were attained, usually within 10 min. Images were recorded in a real-time fashion with a charge-coupled device (CCD) camera at 10- to ~30-s intervals. Background fluorescence from target cells was low compared with fluorescence of Env-expressing cells.

For the Env FP insertion, bis-ANS was also used as the probe, but added at 20 or 35 s after the introduction of target cells. The increased fluorescence intensity was attributed to FP exposure; hence, variation in the delayed time of addition of the probe can be used to evaluate membrane penetration of FP, which blocks bis-ANS attachment to FP. Image processing was performed using MetaMorph software (Carl Zeiss Meditec, Göttingen, Germany).

Preparation of raft-liposomes
The liposomes were prepared as the detergent-resistant lipid-raft (denoted raft-liposomes) with molar ratio 1:1:1 cholesterol-sphingomyelin-phosphatidylcholine (15) , solubilized in a 4:1 mixture of chloroform-methanol. Aliquots of the raft-liposomes (final concentration 5 mM) were evaporated by a stream of nitrogen gas. The remaining chloroform-methanol was removed under vacuum for at least 1 h. Subsequently, the raft-liposome mixture was added with 2% cholesteryl hemisuccinate Tris salt-10% n-dodecyl-β-D-maltopyranoside-10% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) detergent in a ratio of 5:1 as the raft-liposomes/detergent micelle. The micelle was dissolved in 1 ml of solubilization buffer (50 mM Hepes, 5 mM MgCl2, 1 mM CaCl2, and 150 mM NaCl, pH 7.5) and diluted 10-fold in the solubilization buffer before use. The diluted suspension was frozen, thawed, and vortexed at least four times. The homogenized liposomes were prepared by sonication and purification with ultracentrifugation at 20,000 g for 30 min.

L1-SPR
SPR experiments were performed on a BIAcore 3000 biosensor system using an L1 biosensor chip (BIAcore AB, Uppsala, Sweden). The surface of an L1 sensor chip was cleaned with 40 mM octyl-D-glucoside and 50 mM NaCl followed by 30% ethanol. Raft-liposomes/detergent micelles (0.5 mM lipid concentration) were mixed with HXB2 Env (200 nM) by gentle shaking at 4°C. The mixture was immediately injected at a flow rate of 2 µl/min (200 µl) into the chamber. The analyte CD4-X4 mixtures (1 µM–250 nM, 50 µl) or CD4 (1 µM, 50 µl) molecules at a flow rate of 10 µl/min were injected and captured on the HXB2 Env-embedded surface. At the end of a binding measurement, the surface was cleaned by a 10-min injection of 40 mM octyl-D-glucoside and 50 mM NaCl at a flow rate of 10 µl/min. SPR data were analyzed with BIAevaluation 3.0 software (Biacore AB). The kinetic data were fit by the software using a conformation change fitting model. The quality of fit was estimated by calculating {chi}2 values and inspecting residuals. Shedding of gp120 from gp41 can be deduced from the dissociation stage of the sensorgrams. To directly examine the composition, the eluent from the channel cell was collected with the aid of a self-written automated recovery program after CD4-X4 or CD4 engagement and analyzed with Western blotting. The cell content-mixing activity has been shown to be retained (unpublished data) for HXB2 Env incorporated in the raft-liposomes.

Shedding of HXB2 gp120 from gp41 by L1-SPR
Shedding of gp120 from gp41 can also be deduced from the dissociation stage of the sensorgrams. To directly examine the composition, the eluent from the channel cell was collected with the aid of a self-written automated recovery program after CD4-X4 or CD4 engagement and analyzed with Western blotting as described above.

Cell fluorescence image experiments
HXB2 Env-expressing HeLa cells were harvested and attached on glass coverslips. They were washed with PBS and incubated with CD4-X4 or CD4. The cells were then fixed by 4% paraformaldehyde at different times to dissect the kinetics of conformational changes induced by addition 10 µg/ml of CD4-X4 or CD4. The gp41 NC-1 antibody-sensitive conformations (NSCs) were detected by NC-1 antibodies with 3 h of incubation in PBS containing 3% BSA at room temperature. The cells were subsequently washed three times with PBS and incubated with fluorescein isothiocyanate (FITC) -conjugated goat anti-mouse antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 2 h at room temperature and further washed three times with PBS. The coverslips were then mounted with PBS containing glycerol and analyzed by fluorescence microscopy (MetaMorph).

Measurements of kinetics of HIV-1 Env recruitment
For analysis, 5 x 105 HIV-1 Env-enhanced green fluorescent protein (EGFP) -expressing HeLa (effector) cells were adhered to glass coverslips and placed into an open cell chamber. An equal number of CD4-alone- or CD4-X4-expressing NIH3T3 (target) cells were diluted in the medium without serum and subjected to the effector cell-coated coverslips in the open cell chamber. For kinetics studies, the cell were imaged on a real-time basis by a MetaMorph microscope (Carl Zeiss Meditec), and images were recorded on a CCD digital camera. At least several hundred cells were observed. Image processing was performed using MetaMorph software.

Measurements of kinetics of lipid mixing and content mixing
For lipid mixing, HIV-1 Env-expressing (effector) HeLa cells and CD4-X4- or CD4-alone-expressing (target) NIH3T3 cells were incubated, respectively, for 15 min with 20 µM DiO (green) and DiI (red) in RPMI 1640 medium without serum. The cells were then washed three times with medium or PBS, and resuspended at 106 cells/ml in RPMI 1640 medium without serum. DiI-labeled target cells were cocultured with DiO-labeled effector cells at 37°C, and lipid dye mixing was monitored in a real-time fashion. For content mixing, the effector and target cells were labeled, respectively, with calcein AM (green) and 5- and 6-{[(4-chloromethyl)benzoyl]amino} tetramethylrhodamine, red (CMTMR) at concentrations of 10 and 20 µM for 1 h at 37°C. CMTMR-labeled target cells were cocultured with calcein-labeled effector cells at 37°C, and dye redistribution was monitored real time. All of the fluorescence images were monitored by a fluorescence microscope (MetaMorph) coupled to a CCD camera. The dyes were purchased from Molecular Probes (Eugene, OR, USA). At least several hundred cells were observed. Image processing was performed using MetaMorph software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gp41 FP exposure occurred rapidly after CD4-X4 or CD4-alone engagement
According to current fusion models (10 , 18) , CD4 induces conformational changes in the HIV-1 Env, resulting in exposure of the hydrophobic fusion domain of gp41 (19 , 20) . It has been shown that exposure of influenza HA hydrophobic binding sites could be detected by a sharp increase in fluorescence emission of bis-ANS (21) . In Supplemental Fig. 1, it is clearly shown that the major bis-ANS hydrophobic binding resides in FP. Thus, the enhanced intensity of the fluorophore is reduced by ~50% when the FP domain was excluded from a construct containing gp41 ectodomain, gp41 (amino acids 512–534). The result indicates that a significant increase in bis-ANS fluorescent intensity is attributable to binding to the FP region.

Figure 1 A shows only the images at 0-, 0.5-, 1-, and 15-min time points. A movie is accessible as Supplemental Movie 1. The exposure was detectable at ~30 s in the presence of CD4-X4 cells. Similarly, when exposed to the target cells expressing CD4 only, HIV-1 Env-expressing cells showed the same exposure kinetics as CD4-X4 cells (Fig. 1B ; Supplemental Movie 2). Of note is the fact that Env-expressing cells did not noticeably light up in the NIH3T3 cells that do not bear CD4-X4 proteins (Fig. 1C ; Supplemental Movie 3). In the brightfield images (Fig. 1A-C , first panel), we can distinguish between the original Env-expressing cells and the added target cells. In addition, little fluorescence intensity change was observed in the target cells. Figure 1D illustrates changes in the fluorescence intensity of Env effector cells against time after addition of CD4-X4, CD4-alone, or mink CD4-X4 target cells as indicated. Note that the Env effector cells display a rapid exposure of FP with a short lag time (30 s) on CD4-X4 and CD4-alone binding. In contrast, no fluorescence change is found for the Env effector cells with addition of mink CD4-X4 cells (as a negative control).


Figure 1
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  		Figure 1. HIV-1 HXB2 gp41 FP exposure (A–D) and insertion (E–G) by adding bis-ANS fluorescence on engagement of CD4-alone- or CD4-X4-expressing target cells. Fluorescence images (A–C, E–F) are shown in pseudo-color, which represents increasing intensity in the following order: black, purple, blue, green, yellow, red, and white. The fluorescence images were taken from the same fields of Env-expressing HeLa cells (the first brightfield) on addition of CD4-X4 (A), CD4-alone (B), or mink CD4-X4 (C) NIH3T3 cells, respectively. There is a large increase in fluorescence of the HeLa cells at ~30 s after exposure to CD4-X4 and CD4-alone target cells (A, B). There is little or no change in fluorescence of Env-expressing HeLa cells in response to mink CD4-X4-expressing NIH3T3 cells (C). The first panels of A–C are brightfield images on addition of target cells; the second to fifth panels of A–C are fluorescence images at 0-, 0.5-, 1-, and 15-min time points after addition of target cells. Scale bars = 10 µm. D) Change of fluorescence intensity on Env effector cells against time after addition of CD4-X4, CD4-alone, or mink CD4-X4 target cells as indicated. The Env effector cells show a rapid exposure of FP with little lag time (30 s) on CD4-X4 and CD4-alone binding. The Env-expressing effector cells exhibit no fluorescence change after addition of mink CD4-X4 cells. For the HIV-1 Env FP insertion, bis-ANS was added immediately at 20 s (E) and 35 s (F), respectively, after addition of target cells and the fluorescence change was monitored for at least 1 min, the results of which were shown only at the 10-, 30-, and 60-s time points. The first panels of E, F are brightfield images on addition of target cells. The increasing fluorescence intensity was only seen in E (panels 3–5) and confirmed that the FP exposure was at ~30 s. In F, there is no fluorescence intensity increase, indicating that the FP was not exposed outside after 35 s. G) Change in fluorescence intensity on Env effector cells against time after addition of bis-ANS at 20 and 35 s, respectively, after incubation of target cells. Only the Env effector cells after addition of bis-ANS at 20 s show an increased fluorescence. These experiments implied that FP inserted into the membrane immediately after its exposure (<35 s).

gp41 FP inserts into the membrane immediately after its exposure
Delayed addition of bis-ANS was used to probe the membrane insertion of HIV-1 Env FP. According to Fig. 1A, B and Supplemental Movies 1 and 2, FP became exposed at ~30 s; hence, we chose the two time points 20 and 35 s. Bis-ANS was added at 20 s (Fig. 1E ; Supplemental Movie 4) and 35 s (Fig. 1F ; Supplemental Movie 5), respectively, after addition of target cells. The fluorescence change was followed for at least 1 min, the results of which are shown only at 10, 30, and 60 s. The increased fluorescence intensity was only seen in Fig. 1E (panels 3–5), confirming the fact that FP exposure occurred at ~30 s after the Env-receptor-coreceptor interaction. As seen from Fig. 1F , FP was not exposed after a 35-s delay of adding the fluorescence probe. Figure 1G delineates changes in the fluorescence intensity of Env effector cells against time following introduction of bis-ANS at 20 and 35 s after addition of target cells. All of above results support the notion that FP inserts into the membrane immediately after its exposure. The ramifications of the result in the membrane fusion will be elaborated on in the Discussion.

No significant difference in the kinetics of gp41 prehairpin exposure is detected in the presence or absence of X4 on Env-raft-liposomes of L1 surface, but T-649 binding affinity to gp41 is more sensitive to X4
Interactions of Env and CD4 coreceptors trigger a cascade of conformational changes in Env that drive the membrane fusion. The resultant structure of the exposed N- and C-helix regions, termed prehairpin, can be probed by the entry inhibitory peptide T-20 or T-649 (22 23 24 25) . T-20 or T-649 binds HR1 (heptad repeat 1) in gp41 and prevents formation of the six-helix bundle (SHB). Here we used T-649 to investigate the effect of X4 on the exposure of prehairpin. The HXB2 Env-raft-liposomes are immobilized on the L1 sensor chip as described in Materials and Methods. The T-649 peptide was added after CD4-alone or CD4-X4 engagement at different times indicated by arrows in Fig. 2 . The prehairpin was formed rapidly (1 min) on either CD4-alone or CD4-X4 engagement, and the kinetics was in agreement with the cell fluorescence image results (Supplemental Fig. 4). Although binding kinetics was similar with or without the coreceptor (~1 min), binding affinity was substantially different in the SPR measurements. Association of T-649 to the prehairpin in the presence of X4 was characterized by higher binding affinity (ka 8370 vs. 4790 M–1 s–1; KD 75 vs. 99 nM) that may reflect structural alteration of gp41, which was in turn caused by X4 attachment to gp120. Higher binding affinity of T-649 to the gp41 prehairpin on introducing X4 would provide more energy for Env to proceed with further structural changes, for instance, shedding of gp120, formation of the gp41 NSC, or Env recruitment in the membrane, to be examined in the following sections.


Figure 2
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  		Figure 2. Kinetics analysis of T-649 binding to HXB2 gp41 prehairpin on CD4 or CD4-X4 engagement by L1-SPR. Env was immobilized on a L1 chip, CD4 or CD4-X4 at 500 nM was passed over the chip surface for 5 min at a flow rate of 10 µl/min, and T-649 at 5 µM (10 µl/min, 5 min) was injected subsequently at different time course, as indicated by arrow in the sensorgram. All injections were carried out in duplicate. The prehairpin formed rapidly (1 min) on either CD4-alone or CD4-X4 engagement, kinetically in agreement with the cell fluorescence image results (Supplemental Fig. 4). On the other hand, the binding of T-649 to prehairpin in the presence of X4 was characterized by a large binding affinity (ka 8370 vs. 4790 M–1 s–1; KD 75 nM vs. 99 nM), which was assumed to provide more energy for HXB2 Env to proceed with the subsequent structural changes, for instance, shedding of gp120, formation of the gp41 NSC, or Env recruitment in the membrane. RU, response units.

HXB2 gp120 shedding from gp41 was dramatically induced on CD4-X4 addition to the Env complex
HXB2 gp120/gp41 was incorporated on the L1 sensor chip and then CD4 or CD4-X4 was added to monitor the shedding of gp120, another key step in the early fusion reaction, by the SPR sensorgrams (Fig. 3 ). The dissociation curve could result from the unbound analyte (CD4 or CD4-X4). Interestingly, it was observed that dissociation was promoted only for the case of CD4-X4 (kd 10–3 s–1) (Fig. 3B ) engagement but not for CD4-alone (kd 10–5 s–1) (Fig. 3A ) binding. The alternate interpretation is that decreased signals in Fig. 3B arose from shedding of gp120 along with CD4-X4. In other words, compared with CD4 engagement only, the coreceptor induced the dislodgement of gp120 from membrane-anchored Env. To verify the hypothesis we collected the eluent from the channel cell after CD4 or CD4-X4 addition and analyzed it by Western blotting. In Fig. 3C , the gp120 was clearly seen in the presence of X4 but was not seen for CD4-alone engagement. We found that the gp120 shedding occurred immediately after CD4-X4 binding from the dissociation profile of the sensorgram, which leveled off within the first 10 min. In addition, we also verified that gp120 shedding was accompanied by CD4 and X4 (Fig. 3C , CD4-X4/eluent column).


Figure 3
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  		Figure 3. HXB2 gp120 shedding. gp120 shedding was visualized by the dissociation curve of the SPR sensorgrams after CD4 (A) or CD4-X4 (B) binding to HXB2 Env-embedded surface on the L1 sensor chip. CD4 or CD4-X4 at 1 µM was injected over the Env-loaded surface. The refractive index change shown was the correction after subtraction of the blank sensorgram. The dissociation phase of the sensorgram was compared on CD4 or CD4-X4 engagement. The dissociation rate constant (kd) was evaluated using the conformation change fitting model of BIAevaluation 3.0 software. The dissociation curve of the SPR sensorgram was intense (arrow) only in the presence of X4 (kd 10–3 s–1 vs. 10–5 s–1, CD4-alone engagement), which resulted from the shedding of gp120 from gp41. To verify this notion, the eluent from the L1 channel cell after CD4 or CD4-X4 engagement was collected and visualized by Western blotting (C). Lanes 2 and 4 were the eluents after binding by CD4-X4 and CD4-alone, respectively. The Env-raft-liposome layer immobilized on the L1 chip after CD4-X4 or CD4-alone (lanes 1 and 3) engagement was removed by 30 µl of 40 mM octyl-D-glucoside and 50 mM NaCl after the binding sensorgram. The top, middle, and bottom panels were hybridized with antibodies against HXB2 gp120, CD4, and X4, respectively, confirming that gp120 shedding was accompanied by CD4 and X4. RU, response units.

NSC was formed faster in the presence of X4 coreceptor in comparison with CD4-alone engagement
Having investigated the effect of X4 on HXB2 gp120 shedding and the accessibility of gp41 prehairpin, we pursued the putative next process in the cascade of Env conformational changes leading to membrane fusion, namely, the formation of NSC monitored by the cell fluorescence images. Figure 4 demonstrates NSC formation in gp41 detected after incubation for different periods with CD4-alone or CD4-X4 mixtures by NC-1 antibodies. NSC was detected by immunostaining with NC-1 and FITC-conjugated anti-mouse antibodies, indicating the viability of green fluorescence as a probe for gp41 NSC formation. Figure 4 shows only a part of each image, with full-size images being available in Supplemental Fig. 5. In Fig. 4 , the onset of green fluorescence was observed at 10 min on CD4-X4 engagement, and the intensity was enhanced progressively at 20 min and subsequent intervals. In contrast, barely detectable green fluorescence emerged at 20 min and gradually intensified at 30 min on CD4-alone engagement. No green fluorescent signal could be observed within the first 10 min of either CD4 alone or CD4-X4 engagement (data not shown). Thus, the fluorescence imaging data point to the role of coreceptor in accelerating gp41 NSC formation.


Figure 4
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  		Figure 4. Kinetics of HXB2 gp41 NSC formation on CD4 or CD4-X4 engagement. gp41 NSC formation was visualized by NC-1 antibodies. NSC on Env-expressing HeLa cells was visualized by immunostaining with NC-1 mAb and FITC-conjugated anti-mouse antibodies as the secondary antibody. HeLa cells expressing HXB2 Env were incubated for 0, 10, 20, 30, and 40 min as indicated with CD4-alone (left panel) or with the CD4-X4 complex (right panel). The staining procedure is described in Materials and Methods. Full-size images are available in Supplemental Fig. 5. The green fluorescence shows the location of the NSC probed by NC-1 mAb and FITC-conjugated anti-mouse antibodies. The green fluorescence was budding at 10 min on CD4-X4 engagement and increased progressively at 20 min and at subsequent intervals. As a comparison, the green fluorescence picked up intensity at 20 min and gradually enhanced thereafter on CD4-alone engagement. Thus, the fluorescence imaging data support the role of coreceptor in accelerating gp41 NSC formation. Scale bars = 10 µm.

HIV-1 HXB2 Env recruitment occurs only on addition of CD4-X4 target cells after the NSC formation
Previous studies have established the fact that higher order structure of multiple Env-receptor complexes is required to form a fusion pore (26 , 27) . Thus, to examine whether HIV-1 Env is recruited after binding to target cells, we constructed the EGFP fusion protein to probe the movement of HIV-1 HXB2 Env protein on the HeLa effector cell. CD4-alone or CD4-X4 was expressed in NIH3T3 target cells. The recruitment of Env-EGFP on effector cell surface was recorded by a CCD digital camera on addition of target cells. In the real-time experiment, HIV-1 Env-EGFP recruitment was observed after contact with adherent CD4-X4 cells (Fig. 5 A, arrow), although not with adherent CD4-alone cells (Fig. 5B ). Figure 5 showed only 0-, 5-, 13-, and 20-min time points. The image movies are available as Supplemental Movies 6 and 7. Initially, the Env-EGFP was distributed throughout effector cells with no intensive fluorescence clusters found at the interface with CD4-X4 target cells. At ~13 min, onset of the HIV-1 Env-EGFP fluorescence can be observed, which clusters at the initial contact site with target cells, and the intensity continued to increase up to 20min postincubation until complete membrane fusion (as defined by aqueous content mixing), which starts to show up at ~20 min (Fig. 6 C).


Figure 5
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  		Figure 5. Recruitment of HIV-1 Env-EGFP (green) on the effector HeLa cells on engagement of CD4-X4-expressing (A) or CD4-alone-expressing (B) NIH3T3 cells. Fluorescence images were taken from the same fields of Env-EGFP-expressing HeLa cells (the first brightfield) on addition of CD4-X4 (A) and CD4-alone (B) NIH3T3 cells, respectively. Only images at the 0-, 5-, 13-, and 20-min time points are shown. Movie versions are available as Supplemental Movies 6 and 7. HIV-1 Env-EGFP recruitment was observed after contact with adherent CD4-X4 cells and occurred at ~13 min (A, arrow), but not in the presence of CD4-alone target cells (B). Scale bars = 10 µm.


Figure 6
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  		Figure 6. Lipid mixing and content mixing of HIV-1 Env-expressing HeLa cells and target cells. For lipid mixing (A, B), HIV-1 Env-expressing (effector) and CD4-X4- or CD4-alone-expressing (target) NIH3T3 cells were labeled with the 20 µM DiO (green) and DiI (red) probes, respectively. CD4-X4-expressing (A) and CD4-alone-expressing (B) DiI-labeled target cells were cocultured with DiO-labeled effector cells at 37°C, and lipid dye mixing was monitored microscopically as described in Materials and Methods. Only CD4-X4-expressing target cells cocultured with effector cells show the lipid dye mixing at ~15 min, and the intensity continued to increase up to 20 min (arrow). Movie version can be accessed as Supplemental Movies 8 and 9. For content mixing (C, D), HIV-1 Env-expressing HeLa (effector) cells were incubated with CD4-X4 (A) or CD4-alone (B) NIH3T3 (target) cells. CMTMR-labeled target cells (red) were cocultured with calcein-labeled effector cells (green) at 37°C, and dye redistribution was monitored microscopically as described in the Materials and Methods. Content mixing occurred at ~20 min after CD4-X4 (C) target cells binding (arrow), but not in the absence of X4 (D). Movie versions are available as Supplemental Movies 10 and 11. Scale bars = 10 µm.

Cell-cell fusion reaction occurred only on CD4-X4 engagement
Lipid mixing
HIV-1 HXB2 Env-expressing and CD4-X4- or CD4-alone-expressing NIH3T3 cells were probed with DiO (green) and DiI (red), respectively. CD4-X4-expressing (Fig. 6A ) or CD4-alone-expressing (Fig. 6B ) DiI-labeled target cells were cocultured with DiO-labeled effector cells at 37°C, and lipid dye mixing was recorded in real time by a fluorescence microscope coupled to a CCD camera. Only CD4-X4-expressing target cells cocultured with effector cells display lipid dye redistribution at ~15 min, indicating that the X4 coreceptor for HXB2 Env is necessary for the lipid mixing process. Furthermore, lipid mixing occurs almost concomitantly with Env recruitment. The movies for the lipid mixing process are available as Supplemental Movies 8 and 9.

Content mixing
HIV-1 Env-expressing (effector) HeLa cells were labeled with calcein AM, (green) and CD4-X4-expressing (target) NIH3T3 cells were loaded with the cytoplasmic dye CMTMR (red) for 1 h at 37°C. CMTMR-labeled target cells were cocultured with calcein-labeled effector cells at 37°C, and dye redistribution was monitored microscopically by a CCD camera. The results are similar to the lipid mixing data; thus, content mixing was observed only in the CD4-X4-expressing cells cocultured with effector cells at ~20 min postincubation (Fig. 6C ). The result implicates the X4 coreceptor in the important step of fusion pore formation and/or stabilization in the cell fusion cascade. The movies for the cell content mixing can be perused as Supplemental Movies 10 and 11.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Viability of the L1-SPR system in mimicking the HXB2 Env-CD4 interaction
L1 biosensor SPR was performed on a BIAcore 3000 biosensor system. In view of the importance of rafts to HIV-1 function (15 16 17 , 28) , HXB2 gp120/gp41 was embedded in the liposomes containing detergent-resistant rafts as noted in Materials and Methods. The Env-raft-liposomes were then captured by the L1 hydrophobic anchors to investigate the effect of receptor or coreceptor on gp120/gp41 by adding different probes. First, we measured the dissociation constant (KD) of CD4 to the gp120/gp41-raft-liposome complex on the L1 sensor chip. After immobilization of the gp120/gp41-raft-liposomes on the L1 chip to a level that yields a signal of ~5000 response units, each aliquot of increasing CD4 concentrations at 100, 250, 500, and 1000 nM was added (Supplemental Fig. 2). KD was analyzed after subtracting the background using the BIAevaluation 3.0 software. As shown in Supplemental Fig. 2, the KD of CD4 to the gp120/gp41 complex was 5.5 nM, in close agreement with previous studies (29 , 30) . Thus, we demonstrated the viability of this L1-SPR system in mimicking the Env-CD4 interaction on the viral surface.

Coreceptor for HIV-1 Env conformational changes
HIV entry is known to require interaction of the viral Env glycoprotein with CD4 and cognate cellular chemokine receptors. The diverse cell tropism among different isolates was attributed to the differential use of chemokine receptors by HIV (31 , 32) . Although some HIV isolates are CD4-independent strains, the coreceptor is absolutely required (33 34 35 36) , suggesting that the coreceptor in HIV entry into target cells has an essential function. It has been demonstrated that conformational changes triggered on the CD4 engagement are not sufficient to drive fusion (37 38 39) . It is thus likely that coreceptor binding induces further conformational changes in the gp120/gp41-CD4 complex to promote cell fusion by gp41. In the present study, we attempt to dissect mechanistically the role of coreceptors in HIV-1-mediated fusion. From the kinetics and temporal order of the steps on which the coreceptor exerts its influence, we are able to improve our understanding of the Env-mediated fusion reaction.

Initial Env conformational changes include FP exposure, insertion, and prehairpin intermediate formation, which occur at ~30 s to 1 min
Analysis of Fig. 1A--G reveals that bis-ANS fluorescence enhancement persists when the probe is cocultured with CD4 or CD4-X4. A 20-s delay of bis-ANS addition results in little difference, but the enhancement was abolished with a 35-s delay. These observations can be rationalized as follows. First, no fluorescence increase was found with bis-ANS incubated with the target cells, indicating no membrane penetration by the probe alone (Fig. 1C ). As FP was exposed after receptor binding to Env, bis-ANS bound to FP at ~30 s. When FP inserts into the membrane, the bound probe translocates to the hydrophobic milieu along with FP, hence the persistent intensity shown in Fig. 1D . The differential results for the 20- and 35 s delays in the addition of the probe to CD4-X4 or CD4 indicate that, by 35 s after Env engagement to the receptor, FP inserts into the (target) membrane; hence, there is no intensity change for bis-ANS because FP is not available to the probe (Fig. 1E-G ). It is noteworthy that the short lapse (15 s) for the window of access of FP by bis-ANS is consistent with the concept that FP is at least partially sequestered by gp120 when it is protruding toward the target membrane without being totally exposed to the aqueous environment.

According to our results (Fig. 1) , exposure and insertion of the gp41 FP occurred in the initial steps in the HIV-1 fusion reaction. Previous in vitro studies showed that FP insertion into liposomes was complete within 1 min (40 , 41) , which was in line with our results. In our study, the insertion of FP was monitored from the fluorescent intensity change by adding bis-ANS at 20 s (Fig. 1E ) and 35 s (Fig. 1F ) after target cell engagement, indicating that FP inserted into the membrane shortly after its exposure. A previous investigation also pointed out that gp120 interacts with target cell CD4 and undergoes conformational changes leading to exposure of gp41, which then forms a prehairpin intermediate (10) . In this state, FP inserts into the target membrane after its extrusion, by coupling to the conformational alteration in the HR regions, to the contact site (18 , 42) . Incidentally, we are the first to prove the existence of these FP and prehairpin temporal sequences and kinetics on living cells in the presence or absence of X4 molecules.

Figure 2 illustrates kinetically the prehairpin exposure by L1 biosensor. Higher binding affinity of T-649 to the gp41 prehairpin on X4 addition (ka 8370 M–1 s–1; KD 75 nM) would provide more energy for Env to proceed with further structural changes, for instance, shedding of gp120, the gp41 NSC formation, or Env recruitment in the membrane, to be discussed in the following sections. Furthermore, by means of the SPR dissociation curve, the dissociation constants (kd) of T-649 interaction with prehairpin were equally slow (kd 10–4 s–1) either in CD4 or CD4-X4 binding, indicating that the Env HR1 region has strong affinity to T-649 inhibitor peptide.

It is noteworthy that binding of the prehairpin intermediate by T-649 occurs in 1 min after CD4 incubation with the envelope protein. Recent studies have suggested that NC-1 antibodies could recognize both a SHB and N-helix trimer (43 , 44) , and the exposed intermediate conformation may coexist with the SHB. It was reported that the prehairpin could directly participate in pore formation before SHB is formed (43) . Therefore, membrane apposition induced in the course of SHB formation assists the extended helix bundle to exert its fusogenic activity. Inhibition of fusion by synthetic C-helix peptides (e.g., T-649, C34, and T-20) can be achieved either by inhibiting the formation of the SHB (18 , 56) , which prevents membrane apposition, and/or by directly binding the N-helix and inhibiting its fusogenic function (43) . The results are compatible with our kinetic observation that the T-649 recognition of the prehairpin well precedes the formation of NSC (at ~10 min) (Fig. 4) .

gp120 shedding from gp41 occurred after FP exposure and prehairpin formation, but before NSC formation
Another important early event of Env conformational change is gp120 dissociation from gp41. Although some studies indicated that the gp120 dissociation (45 46 47) could occur after surface CD4 (sCD4) -alone binding, Blumenthal and coworkers (11) pointed out that the sCD4 is more accessible when interacting with gp120/gp41 in comparison with the membrane-anchored CD4 and hence could induce extraneous conformational change resulting in improper or premature shedding of gp120. In the present work, we used the cell membrane-anchored CD4 in lieu of sCD4. The apparent dissociation was seen by SPR in CD4-X4 engagement but not in the absence of X4 (Fig. 3) . That shedding of gp120 contributes to the dissociation phase observed in the SPR sensorgram was confirmed by comparing the eluents from the channel chip after CD4 or CD4-X4 docking to gp120/gp41 on the L1 sensor chip and visualized by Western blot assay (Fig. 3C ). Interestingly, the shedding of gp120 from gp41 occurs immediately (~1 min) on the membrane-anchored CD4-X4 engagement and continues throughout the first 10 min (Fig. 3B ). The gp120 dissociation kinetics shown herein is similar to that in the study by Moore et al. (48) using sCD4. The gp120 dissociation rate constant (kd) on CD4-X4 engagement (Fig. 3B ) was faster than that for CD4-alone (Fig. 3A ) binding (kd 10–3 vs. 10–5 s–1).

Furthermore, gp120 shedding in the presence of X4 may also explain the accelerated NSC formation in the presence of X4 (to be discussed later). Therefore, the coreceptor engagement appears to provide additional energy to promote gp120 shedding in the early stage of Env conformational changes. It is also in agreement with the notion that the uncleaved gp120/gp41 cannot trigger the cell-cell fusion reaction (11) .

gp120 shedding facilitates formation of the NC-1-cognate conformation
We next attempt to address the role of coreceptor in the formation of a SHB. Biophysical and structural analyses have indicated that the N- and C-helical regions associate to form a highly stable SHB, in which a central trimeric coiled coil formed by the N-terminal helical region of gp41 is surrounded by three antiparallel C-terminal helices that bind to conserved hydrophobic grooves on the coiled coil surface (49 50 51 52) , considered as a crucial step of the HIV-1 fusion reaction leading to the fusion pore formation. SHB was deduced from the crystallographic study of the gp41 core structure (49 , 51 , 53 , 54) , and its formation was suggested to drive the apposition of the target and viral membranes. During the replication cycle of HIV-1, Env molecules oligomerize in the endoplasmic reticulum and are transported to the Golgi apparatus for processing by the protease; hence, the native HIV-1 Env molecules located on the surface of the virion or the infected cell are displayed as oligomeric gp120/gp41 complexes (55) . The conformation-specific monoclonal antibody (mAb) NC-1 that was induced by SHB modeled with a N36(L6)C34 polypeptide (56) has been used for detecting SHB formation by the gp120/gp41 oligomer (57 58 59) . However, the exact conformation recognized by NC-1 is still under debate (44 , 60) . Therefore, the term NC-1 antibody-sensitive conformation (NSC), instead of SHB, is used in the present work. Of note, we observed that membrane-anchored CD4-X4 binding to HXB2 Env-expressing cells facilitated gp41 NSC formation at 10 min, whereas for the case of CD4-alone the formation requires a longer lag time and takes place at ~20 min. This observation is at odds with the concept that SHB is the conformation recognized by NC-1 because we found the onset of lipid mixing at ~13 min, later than the NSC formation time (10 min); yet SHB formation was thought to occur at the time of pore formation or dilation (61 , 62) . No gp41 NSC formation was detected within the first 10 min of Env-receptor (-coreceptor) association (data not shown) with or without the presence of X4. In this context, it is reasonable to note that, in the presence of X4, shedding of gp120 from gp41 may contribute to the accelerated gp41 NSC formation (i.e., the unclamped gp41 oligomer facilitates the approach of NC-1), as X4 is required for gp120 shedding. Although HXB2 gp41 NSC formation could take place on either CD4-X4 or CD4-alone engagement, the lack of X4 could abrogate the complete cell-cell fusion (Fig. 6D ), in agreement with previous findings (11 , 63 64 65) . Comparison of the binding constant of NC-1 to Env with and without X4 revealed that X4 engagement enhanced the interaction of the antibody with its ligand (ka values for Env-NC-1 are 5770 and 2400 M–1 s–1 with and without X4, respectively (by L1-SPR, data not shown), suggesting structural alteration indirectly induced by X4. CD spectroscopic analysis also concurs with this assertion (Supplemental Fig. 3). This X4-induced structural change may account partially for the failure of the virus to mediate complete cell-cell fusion in the absence of the coreceptor. Because the fluorescence intensity monitoring NSC continued to rise over a period of several minutes after its onset at 10 min after incubation of Env and CD4-X4, it is possible that NSC is an intermediate structure in the process leading to SHB. This concept is consistent with the conclusion drawn from a temperature jump study that completion of SHB formation takes place immediately after the pore formation (61) , considering our view that SHB follows NSC (at ~10 min) and the content mixing occurs at 13 min.

Functional implications of gp120 shedding
In an early mutational study on correlation of gp120 shedding with the Env fusion activity (66) , some of the mutants with amino acid changes in the C3 and C4 regions (368 D/P, 382 F/L, 420 I/R, 433 A/L, and 438 P/R) exhibited reduced shedding of gp120 after soluble CD4 incubation. These mutants also had a greatly impaired syncytium-forming capability. Analysis of the positions of the mutation revealed, however, that the mutations affected the coreceptor binding (67) . either directly (e.g., 420 I/R and 438 P/R) or indirectly through altered CD4 binding (e.g., 257 T/R and 368 D/P). Some other mutations, e.g., 313 P/S and 314 G/Q indeed displayed a wild-type level of gp120 shedding yet a varying effect on syncytium formation. The results suggested that gp120 shedding greatly facilitated, but was not sufficient to drive, the progress of subsequent steps in the fusion cascade. This is reasonable because the bulky gp120-CD4-coreceptor complex inevitably presents a steric hindrance to gp41 interaction with the target membrane recruitment and refolding near the fusing site. Another investigation on the effect of gp120 mutations (68) indicated a correlation between the infectivity and gp120 shedding for the 429 G/R (427 in the original designation) mutant, consistent with our interpretation, because residue 429 participates in the CD4 binding (69) , mutation of which therefore modulates the coreceptor attachment and hence the gp120 shedding, leading to enhanced infectivity. Combining these results with our findings, we concluded that, for CD4-dependent X4 strains, timely gp120 shedding promoted by coreceptor engagement to gp120—to activate the metastable gp41 and allow the physical approach of two fusing membranes—ushers in subsequent fusion processes.

Because gp120 is noncovalently bound to gp41 on the viral surface, it has been proposed that a disulfide bond-stabilized gp120/gp41 complex is a better antigenic mimic of the trimeric, virion-associated fusion protein structure (70) . Thus, the native structure of the glycoprotein maintained by gp120-clamped gp41 may be necessary for more effective immunogens.

X4 coreceptor contributed to HIV-1 HXB2 Env recruitment concomitant with lipid mixing after NSC formation and leading to the final content mixing
Oligomerization and clustering of the transmembrane protein of HIV and influenza virus have been deduced or suggested to be critical steps in the fusion reaction. Thus, White and coworkers (71) reported that the clustering of influenza hemagglutinin trimers was required for fusion. A previous study demonstrated that HIV-1 virion can be recruited to sites of cell contact in the effector dendritic cells (72) . It was proposed that contact between the effector and target cells facilitates transmission of HIV-1 by locally concentrating virus during the formation of a synapse, through which rapid spread of HIV-1 (73) and protection of the virus from the humoral immune response can be effected (28) . However, the temporal relation to other steps and the mechanism of the Env aggregation have not been elucidated. In this study, we demonstrated that the coreceptor X4 plays a significant role to induce the clustering of HIV-1 HXB2 Env on the effector cells, as the clustering event was not observed in the absence of X4 (Fig. 5B ). Visualization of coreceptor-induced clustering of Env corroborates the proposition that a higher level of multiple Env-coreceptor complexes is necessary to form a fusion pore (27) . Furthermore, Env recruitment induced by CD4-X4-expressing cells was observed at ~13 min, which was concurrent with the lipid mixing step (Fig. 6A ) and subsequent to the NSC formation. It has been reported that inhibition of SHB formation resulted in aborted lipid mixing (74) , in agreement with our finding that NSC formation occurred shortly before the lipid mixing. From Fig. 5 , HIV-1 Env-EGFP fluorescence started to cluster to the initial site of contact with target cells at ~13 min and gradually increased up to 20 min at which full content mixing commenced (Fig. 6C ). To the best of our knowledge, the result shown in Fig. 6A, B is the first report to implicate the coreceptor in the lipid mixing process in the HIV-1-mediated fusion. It is noteworthy that Melikyan et al. (62) have shown that the pore enlargement is rapid and reaches the maximum within the first 20 s after pore opening. Hence it is likely that the Env recruitment along with lipid mixing is a rate-limiting step in the cascade of the Env-mediated fusion process.

In summary, we discovered that the shedding is promoted by the coreceptor X4 for the HXB2 HIV-1 strain used. It is clear that the X4-dependent processes including lipid and content mixing; Env recruitment as well as X4-accelerated NSC formation take place after gp120 shedding.

A refined model on the temporal sequence of CD4-coreceptor induced conformational changes of HIV-1 Env protein
Because the coreceptor is absolutely required for HIV-1 Env-mediated fusion, we sought to elucidate the functional role of X4 in the different stages of fusion event with emphasis on the kinetic aspect to dissect their temporal order and to gain insight into the membrane fusion mechanism. Attachment of the receptor and coreceptor initiates a series of conformational alteration in Env, including extension of FP, insertion of FP into the target membrane, dissociation of oligomeric gp120, gp120 shedding from gp41, refolding of HR1 and HR2, NSC and SHB formation, lipid mixing, and content mixing. In addition, recruitment of the trimeric Env subunits on the membrane surface to cooperate to form a functional fusion pore is also regarded as an essential step. Our results can be summarized by a model depicted in Fig. 7 , describing the time course of important steps in the Env-mediated fusion based on kinetic measurements on these steps after CD4-X4 engagement. The model could be applied to the mechanism of other class I fusion protein-mediated membrane fusion.


Figure 7
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  		Figure 7. A schematic illustration of updated HIV-1 Env-mediated fusion model. The temporal sequence of Env conformational changes in the presence of CD4-coreceptor molecules is as follows: 1) gp120 interaction with CD4; 2) conformational alteration in both molecules to trigger binding to the cognate coreceptor; 3) FP exposure and membrane insertion and prehairpin intermediate formation; 4) gradual shedding of gp120 from the gp41 anchor, further facilitating refolding of HR1 and HR2 of gp41; leading to 5) formation of NSC—an intermediate bundle structure of gp41 core; 6) Env recruitment concomitant with outer leaflet lipid mixing to act in concert for promote and stabilize fusion pore; and 7) full fusion (coalescence of both leaflets of apposing membranes) leading to content mixing.

Because sensitivity of HIV-1 to fusion inhibitors was shown to correlate with the fusion kinetics and Env-coreceptor affinity (65) , we believe a thorough study of individual steps of the fusion process and their temporal relation may also be valuable in understanding the mechanism of drug resistance and assessing the efficacy of fusion-inhibitory therapeutics. As the kinetics of fusion between R5-tropic strains and the target cells may be substantially different from that between X4-tropic strains (75) , a comparative study of the effect of CCR5 on the various stages of fusion reaction is ongoing in our laboratory.


   ACKNOWLEDGMENTS
 
We are grateful to Institute of BioMedical Sciences of Academia Sinica for the fluorescent microscopy assistance. We thank Dr. S. S. Chen for cell lines 293T and for HXB2 gp160-pSVE7, CD4-pcDNA3, pEGFP-N1 plasmids, and antibodies thereof. We also thank the U.S. National Institutes of Health AIDS Research and Reference Reagent Program for the HeLa cells, NIH3T3 cells, X4 plasmids (fusin-pcDNA1), and X4 antibodies. Moreover, we appreciate the help of S. F. Cheng, C. H. Lin, and C. C. Chang with the supplemental data. This work was supported in part by the National Science Foundation and Academia Sinica of the Republic of China.

Received for publication September 12, 2007. Accepted for publication October 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Grewe, C., Beck, A., Gelderblom, H. R. (1990) HIV: early virus-cell interactions. J. Acquir. Immune Defic. Syndr. 3,965-974[Medline]
  2. Center, R. J., Leapman, R. D., Lebowitz, J., Arthur, L. O., Earl, P. L., Moss, B. (2002) Oligomeric structure of the human immunodeficiency virus type 1 envelope protein on the virion surface. J. Virol. 76,7863-7867[Abstract/Free Full Text]
  3. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A., Axel, R. (1986) The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47,333-348[CrossRef][Medline]
  4. Rizzuto, C. D., Wyatt, R., Hernandez-Ramos, N., Sun, Y., Kwong, P. D., Hendrickson, W. A., Sodroski, J. (1998) A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 280,1949-1953[Abstract/Free Full Text]
  5. Sullivan, N., Sun, Y., Binley, J., Lee, J., Barbas, C. F., 3rd, Parren, P. W., Burton, D. R., Sodroski, J. (1998) Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J. Virol. 72,6332-6338[Abstract/Free Full Text]
  6. Lapham, C. K., Ouyang, J., Chandrasekhar, B., Nguyen, N. Y., Dimitrov, D. S., Golding, H. (1996) Evidence for cell-surface association between fusin and the CD4-gp120 complex in human cell lines. Science 274,602-605[Abstract/Free Full Text]
  7. Salzwedel, K., Smith, E. D., Dey, B., Berger, E. A. (2000) Sequential CD4-coreceptor interactions in human immunodeficiency virus type 1 Env function: soluble CD4 activates Env for coreceptor-dependent fusion and reveals blocking activities of antibodies against cryptic conserved epitopes on gp120. J. Virol. 74,326-333[Abstract/Free Full Text]
  8. Trkola, A., Dragic, T., Arthos, J., Binley, J. M., Olson, W. C., Allaway, G. P., Cheng-Mayer, C., Robinson, J., Maddon, P. J., Moore, J. P. (1996) CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 384,184-187[CrossRef][Medline]
  9. Wu, L., Gerard, N. P., Wyatt, R., Choe, H., Parolin, C., Ruffing, N., Borsetti, A., Cardoso, A. A., Desjardin, E., Newman, W., Gerard, C., Sodroski, J. (1996) CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384,179-183[CrossRef][Medline]
  10. Furuta, R. A., Wild, C. T., Weng, Y., Weiss, C. D. (1998) Capture of an early fusion-active conformation of HIV-1 gp41. Nat. Struct. Biol. 5,276-279[CrossRef][Medline]
  11. Jones, P. L. T. J., Korte, T., Blumenthal, R. (1998) Conformational changes in cell surface HIV-1 envelope glycoproteins are triggered by cooperation between cell surface CD4 and co-receptors. J. Biol. Chem. 273,404-409[Abstract/Free Full Text]
  12. Park, E. J., Gorny, M. K., Zolla-Pazner, S., Quinnan, G. V., Jr (2000) A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex. J. Virol. 74,4183-4191[Abstract/Free Full Text]
  13. Finnegan, C. M., Berg, W., Lewis, G. K., DeVico, A. L. (2001) Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion. J. Virol. 75,11096-11105[Abstract/Free Full Text]
  14. Wild, C., Oas, T., McDanal, C., Bolognesi, D., Matthews, T. (1992) A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc. Natl. Acad. Sci. U. S. A. 89,10537-10541[Abstract/Free Full Text]
  15. Aloia, R. C., Tian, H., Jensen, F. C. (1993) Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes. Proc. Natl. Acad. Sci. U. S. A. 90,5181-5185[Abstract/Free Full Text]
  16. Brugger, B., Glass, B., Haberkant, P., Leibrecht, I., Wieland, F. T., Krausslich, H.-G. (2006) The HIV lipidome: a raft with an unusual composition. Proc. Natl. Acad. Sci. U. S. A. 103,2641-2646[Abstract/Free Full Text]
  17. Haque, M. E., Koppaka, V., Axelsen, P. H., Lentz, B. R. (2005) Properties and structures of the influenza and HIV fusion peptides on lipid membranes: implications for a role in fusion. Biophys. J. 89,3183-3194[CrossRef][Medline]
  18. Chan, D. C., Kim, P. S. (1998) HIV entry and its inhibition. Cell 93,681-684[CrossRef][Medline]
  19. Matthews, T. J., Wild, C., Chen, C. H., Bolognesi, D. P., Greenberg, M. L. (1994) Structural rearrangements in the transmembrane glycoprotein after receptor binding. Immunol. Rev. 140,93-104[CrossRef][Medline]
  20. Sattentau, Q. J., Moore, J. P. (1993) The role of CD4 in HIV binding and entry. Philos. Trans. R. Soc. Lond. B Biol. Sci. 342,59-66[Medline]
  21. Korte, T., Herrmann, A. (1994) pH-dependent binding of the fluorophore bis-ANS to influenza virus reflects the conformational change of hemagglutinin. Eur. Biophys. J. 23,105-113[Medline]
  22. Chan, D. C., Chutkowski, C. T., Kim, P. S. (1998) Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target. Proc. Natl. Acad. Sci. U. S. A. 95,15613-15617[Abstract/Free Full Text]
  23. Chen, C. H., Matthews, T. J., McDanal, C. B., Bolognesi, D. P., Greenberg, M. L. (1995) A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion. J. Virol. 69,3771-3777[Abstract/Free Full Text]
  24. Neurath, A. R., Strick, N., Jiang, S. (1992) Synthetic peptides and anti-peptide antibodies as probes to study interdomain interactions involved in virus assembly: the envelope of the human immunodeficiency virus (HIV-1). Virology 188,1-13[CrossRef][Medline]
  25. Wild, C., Greenwell, T., Matthews, T. (1993) A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res. Hum. Retroviruses 9,1051-1053[Medline]
  26. Doms, R. W. (2000) Beyond receptor expression: the influence of receptor conformation, density, and affinity in HIV-1 infection. Virology 276,229-237[CrossRef][Medline]
  27. Kuhmann, S. E., Platt, E. J., Kozak, S. L., Kabat, D. (2000) Cooperation of multiple CCR5 coreceptors is required for infections by human immunodeficiency virus type 1. J. Virol. 74,7005-7015[Abstract/Free Full Text]
  28. Jolly, C., Sattentau, Q. J. (2005) Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity. J. Virol. 79,12088-12094[Abstract/Free Full Text]
  29. Lasky, L. A., Nakamura, G., Smith, D. H., Fennie, C., Shimasaki, C., Patzer, E., Berman, P., Gregory, T., Capon, D. J. (1987) Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor. Cell 50,975-985[CrossRef][Medline]
  30. Schnittman, S. M., Lane, H. C., Roth, J., Burrows, A., Folks, T. M., Kehrl, J. H., Koenig, S., Berman, P., Fauci, A. S. (1988) Characterization of GP120 binding to CD4 and an assay that measures ability of sera to inhibit this binding. J. Immunol. 141,4181-4186[Abstract]
  31. Berger, E. A. (1997) HIV entry and tropism: the chemokine receptor connection. AIDS 11(Suppl A),S3-S16[Medline]
  32. Hoffman, T. L., Doms, R. W. (1998) Chemokines and coreceptors in HIV/SIV-host interactions. AIDS 12(Suppl A),S17-S26
  33. Dumonceaux, J., Nisole, S., Chanel, C., Quivet, L., Amara, A., Baleux, F., Briand, P., Hazan, U. (1998) Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype. J. Virol. 72,512-519[Abstract/Free Full Text]
  34. Endres, M. J., Clapham, P. R., Marsh, M., Ahuja, M., Turner, J. D., McKnight, A., Thomas, J. F., Stoebenau-Haggarty, B., Choe, S., Vance, P. J., Wells, T. N., Power, C. A., Sutterwala, S. S., Doms, R. W., Landau, N. R., Hoxie, J. A. (1996) CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87,745-756[CrossRef][Medline]
  35. Iyengar, S., Schwartz, D. H., Clements, J. E., Hildreth, J. E. (2000) CD4-independent, CCR5-dependent simian immunodeficiency virus infection and chemotaxis of human cells. J. Virol. 74,6720-6724[Abstract/Free Full Text]
  36. Reeves, J. D., Schulz, T. F. (1997) The CD4-independent tropism of human immunodeficiency virus type 2 involves several regions of the envelope protein and correlates with a reduced activation threshold for envelope-mediated fusion. J. Virol. 71,1453-1465[Abstract/Free Full Text]
  37. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., Landau, N. R. (1996) Identification of a major co-receptor for primary isolates of HIV-1. Nature 381,661-666[CrossRef][Medline]
  38. Feng, Y., Broder, C. C., Kennedy, P. E., Berger, E. A. (1996) HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272,872-877[Abstract]
  39. Nagasawa, T., Nakajima, T., Tachibana, K., Iizasa, H., Bleul, C. C., Yoshie, O., Matsushima, K., Yoshida, N., Springer, T. A., Kishimoto, T. (1996) Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc. Natl. Acad. Sci. U. S. A. 93,14726-14729[Abstract/Free Full Text]
  40. Buzon, V., Padros, E., Cladera, J. (2005) Interaction of fusion peptides from HIV gp41 with membranes: a time-resolved membrane binding, lipid mixing, and structural study. Biochemistry 44,13354-13364[CrossRef][Medline]
  41. Saez-Cirion, A., Nir, S., Lorizate, M., Agirre, A., Cruz, A., Perez-Gil, J., Nieva, J. L. (2002) Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring. J. Biol. Chem. 277,21776-21785[Abstract/Free Full Text]
  42. Dimitrov, A. S., Rawat, S. S., Jiang, S., Blumenthal, R. (2003) Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion. Biochemistry 42,14150-14158[CrossRef][Medline]
  43. Korazim, O., Sackett, K., Shai, Y. (2006) Functional and structural characterization of HIV-1 gp41 ectodomain regions in phospholipid membranes suggests that the fusion-active conformation is extended. J. Mol. Biol. 364,1103-1117[CrossRef][Medline]
  44. Dimitrov, A. S., Louis, J. M., Bewley, C. A., Clore, G. M., Blumenthal, R. (2005) Conformational changes in HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion and inactivation. Biochemistry 44,12471-12479[CrossRef][Medline]
  45. Cao, J., Bergeron, L., Helseth, E., Thali, M., Repke, H., Sodroski, J. (1993) Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein. J. Virol. 67,2747-2755[Abstract/Free Full Text]
  46. Fu, Y. K., Hart, T. K., Jonak, Z. L., Bugelski, P. J. (1993) Physicochemical dissociation of CD4-mediated syncytium formation and shedding of human immunodeficiency virus type 1 gp120. J. Virol. 67,3818-3825[Abstract/Free Full Text]
  47. Leavitt, M., Park, E. J., Sidorov, I. A., Dimitrov, D. S., Quinnan, G. V., Jr (2003) Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120. J. Virol. 77,560-570[CrossRef][Medline]
  48. Moore, J. P., McKeating, J. A., Weiss, R. A., Sattentau, Q. J. (1990) Dissociation of gp120 from HIV-1 virions induced by soluble CD4. Science 250,1139-1142[Abstract/Free Full Text]
  49. Chan, D. C., Fass, D., Berger, J. M., Kim, P. S. (1997) Core structure of gp41 from the HIV envelope glycoprotein. Cell 89,263-273[CrossRef][Medline]
  50. Lu, M., Blacklow, S. C., Kim, P. S. (1995) A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat. Struct. Biol. 2,1075-1082[CrossRef][Medline]
  51. Tan, K., Liu, J., Wang, J., Shen, S., Lu, M. (1997) Atomic structure of a thermostable subdomain of HIV-1 gp41. Proc. Natl. Acad. Sci. U. S. A. 94,12303-12308[Abstract/Free Full Text]
  52. Weng, Y., Weiss, C. D. (1998) Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41. J. Virol. 72,9676-9682[Abstract/Free Full Text]
  53. Caffrey, M., Cai, M., Kaufman, J., Stahl, S. J., Wingfield, P. T., Covell, D. G., Gronenborn, A. M., Clore, G. M. (1998) Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41. EMBO J. 17,4572-4584[CrossRef][Medline]
  54. Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., Wiley, D. C. (1997) Atomic structure of the ectodomain from HIV-1 gp41. Nature 387,426-430[CrossRef][Medline]
  55. Salzwedel, K., Berger, E. A. (2000) Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41. Proc. Natl. Acad. Sci. U. S. A. 97,12794-12799[Abstract/Free Full Text]
  56. Jiang, S., Lin, K., Lu, M. (1998) A conformation-specific monoclonal antibody reacting with fusion-active gp41 from the human immunodeficiency virus type 1 envelope glycoprotein. J. Virol. 72,10213-10217[Abstract/Free Full Text]
  57. de Rosny, E., Vassell, R., Jiang, S., Kunert, R., Weiss, C. D. (2004) Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J. Virol. 78,2627-2631[Abstract/Free Full Text]
  58. Yang, X., Farzan, M., Wyatt, R., Sodroski, J. (2000) Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 74,5716-5725[Abstract/Free Full Text]
  59. Yang, X., Lee, J., Mahony, E. M., Kwong, P. D., Wyatt, R., Sodroski, J. (2002) Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin. J. Virol. 76,4634-4642[Abstract/Free Full Text]
  60. Sackett, K., Wexler-Cohen, Y., Shai, Y. (2006) Characterization of the HIV N-terminal fusion peptide-containing region in context of key gp41 fusion conformations. J. Biol. Chem. 281,21755-21762[Abstract/Free Full Text]
  61. Markosyan, R. M., Cohen, F. S., Melikyan, G. B. (2003) HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation. Mol. Biol. Cell 14,926-938[Abstract/Free Full Text]
  62. Melikyan, G. B., Markosyan, R. M., Hemmati, H., Delmedico, M. K., Lambert, D. M., Cohen, F. S. (2000) Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J. Cell Biol. 151,413-423[Abstract/Free Full Text]
  63. Gallo, S. A., Puri, A., Blumenthal, R. (2001) HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process. Biochemistry 40,12231-12236[CrossRef][Medline]
  64. Mkrtchyan, S. R., Markosyan, R. M., Eadon, M. T., Moore, J. P., Melikyan, G. B., Cohen, F. S. (2005) Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion. J. Virol. 79,11161-11169[Abstract/Free Full Text]
  65. Reeves, J. D., Gallo, S. A., Ahmad, N., Miamidian, J. L., Harvey, P. E., Sharron, M., Pohlmann, S., Sfakianos, J. N., Derdeyn, C. A., Blumenthal, R., Hunter, E., Doms, R. W. (2002) Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc. Natl. Acad. Sci. U. S. A. 99,16249-16254[Abstract/Free Full Text]
  66. Thali, M., Furman, C., Helseth, E., Repke, H., Sodroski, J. (1992) Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events. J. Virol. 66,5516-5524[Abstract/Free Full Text]
  67. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., Hendrickson, W. A. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393,648-659[CrossRef][Medline]
  68. Willey, R. L., Martin, M. A., Peden, K. W. (1994) Increase in soluble CD4 binding to and CD4-induced dissociation of gp120 from virions correlates with infectivity of human immunodeficiency virus type 1. J. Virol. 68,1029-1039[Abstract/Free Full Text]
  69. Olshevsky, U., Helseth, E., Furman, C., Li, J., Haseltine, W., Sodroski, J. (1990) Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding. J. Virol. 64,5701-5707[Abstract/Free Full Text]
  70. Binley, J. M., Sanders, R. W., Clas, B., Schuelke, N., Master, A., Guo, Y., Kajumo, F., Anselma, D. J., Maddon, P. J., Olson, W. C., Moore, J. P. (2000) A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74,627-643[Abstract/Free Full Text]
  71. Danieli, T., Pelletier, S. L., Henis, Y. I., White, J. M. (1996) Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J. Cell Biol. 133,559-569[Abstract/Free Full Text]
  72. McDonald, D., Wu, L., Bohks, S. M., KewalRamani, V. N., Unutmaz, D., Hope, T. J. (2003) Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science 300,1295-1297[Abstract/Free Full Text]
  73. Brenchley, J. M., Schacker, T. W., Ruff, L. E., Price, D. A., Taylor, J. H., Beilman, G. J., Nguyen, P. L., Khoruts, A., Larson, M., Haase, A. T., Douek, D. C. (2004) CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp. Med. 200,749-759[Abstract/Free Full Text]
  74. Markosyan, R. M., Cohen, F. S., Melikyan, G. B. (2005) Time-resolved imaging of HIV-1 Env-mediated lipid and content mixing between a single virion and cell membrane. Mol. Biol. Cell 16,5502-5513[Abstract/Free Full Text]
  75. Derdeyn, C. A., Decker, J. M., Sfakianos, J. N., Wu, X., O'Brien, W. A., Ratner, L., Kappes, J. C., Shaw, G. M., Hunter, E. (2000) Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J. Virol. 74,8358-8367[Abstract/Free Full Text]




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