Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M703485200 on May 25, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23104-23116, August 10, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/32/23104    most recent
M703485200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bellamy-McIntyre, A. K.
Right arrow Articles by Poumbourios, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bellamy-McIntyre, A. K.
Right arrow Articles by Poumbourios, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functional Links between the Fusion Peptide-proximal Polar Segment and Membrane-proximal Region of Human Immunodeficiency Virus gp41 in Distinct Phases of Membrane Fusion*Formula

Anna K. Bellamy-McIntyre{ddagger}§1, Chan-Sien Lay{ddagger}1, Séverine Baär||2, Anne L. Maerz{ddagger}, Gert H. Talbo**, Heidi E. Drummer{ddagger}§, and Pantelis Poumbourios{ddagger}3

From the {ddagger}Macfarlane Burnet Institute for Medical Research and Public Health, Prahran, Victoria 3004, Australia, the §Department of Microbiology, Monash University, Clayton, Victoria 3070, Australia, the Department of Microbiology and Immunology, the University of Melbourne, Parkville, Victoria 3058, Australia, the ||Department of Cell Biology, Institut Cochin, Paris, France 75014, and **Proteomics Centre, Baker Heart Research Institute, Melbourne, Victoria 3004, Australia

Received for publication, April 26, 2007 , and in revised form, May 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp666, Trp672, Phe673, and Ile675 in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by ~10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu537 with Trp666, Trp672, Phe673, or Ile675 abolished entry with 50–80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an ~80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The envelope glycoprotein complex (Env)4 of human immunodeficiency virus type 1 (HIV-1) comprises a trimer of receptor binding gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits on the surface of infected cells and virions. Viral entry is initiated when gp120 binds to cell-surface CD4 molecules, inducing structural changes within the gp120 core domain, and leading to the formation of the binding site for the chemokine co-receptors, CCR5 and/or CXCR4 (1–4). gp120 comprises 5 variable loops (V1–V5) that exist outside the gp120 core; V3, and to a lesser degree V1 and V2, contribute to chemokine receptor binding specificity (5–7). The sequential binding of gp120 to CD4 and chemokine receptor triggers the refolding of gp41 into a trimer of hairpins, which mediates membrane fusion (8, 9). gp41 is a class I fusion glycoprotein, being structurally homologous to the fusion glycoproteins of other retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and coronaviruses. The gp41 ectodomain is comprised of an N-terminal fusion peptide, connected through a flexible polar segment to a coiled coil-forming amphipathic {alpha}-helix (N-helix), a centrally located disulfide-bonded loop, a C-terminal amphipathic {alpha}-helix (C-helix), and a membrane-proximal tryptophan-rich region (MPR) (10–16). The ectodomain is anchored to the viral envelope by a C-terminally located transmembrane domain (TMD), which precedes an ~150-residue cytoplasmic domain.

The majority of the gp41 ectodomain appears to be buried by the gp120 trimer. This model for gp41 in the context of prefusion Env is based on the findings that the epitopes of monoclonal antibodies (mAbs) encompassing the fusion peptide and polar segment (residues 521–538), disulfide bonded region (579–613), and C-helix (644–663), are largely occluded in prefusion Env but become exposed following interaction between gp120 and recombinant soluble CD4 (sCD4) or receptor expressing cells (17–20). By contrast, the epitopes of mAbs 2F5 and 4E10 within the MPR (662–667) are exposed in gp120-gp41 prior to CD4 binding indicating an external location for this region (17, 20, 21). These data are largely reflected in a structural model of simian immunodeficiency virus (SIV) Env derived by cryoelectron tomography (22). In this model, a large globular domain comprises a gp120 trimer surrounding the gp41 ectodomain and is anchored to the plasma membrane by a splayed tripod, which corresponds to the MPR in an extensive interaction with the viral envelope. However, an alternative cryoelectron tomographic model of SIV gp120-gp41 revealed a more compact head domain linked to the membrane via a stalk with no evidence of a tripod-like structure (23). The structure of the MPR in situ therefore remains an open question.

gp120-receptor interactions trigger the membrane fusion cascade, beginning with insertion of the fusion peptide into the outer leaflet of the target membrane and gp41 adopting a pre-hairpin intermediate conformation that bridges the viral and cellular membranes (24–26). In the prehairpin intermediate, the trimeric coiled coil of N-helices is exposed and available for interaction with peptides derived from the C-helix that can prevent subsequent stages of the fusion cascade. Likewise, exposed C-helices are available for interaction with synthetic analogues of the N-helix, which also inhibit fusion (18, 19, 27–29). These interactions mimic the antiparallel packing of native C-helices into hydrophobic grooves on the exterior of the coiled-coil that forms the 6-helix bundle (6HB) core, bringing together the N- and C-terminal membrane inserted ends of gp41 (the fusion peptide and TMD), and the associated viral and cellular membranes for merger (30–34). Three phases of cell-cell and virus-cell membrane fusion have been discerned experimentally for class I fusion proteins: lipid mixing or hemifusion; the opening of a small pore through which small solutes can pass; and pore expansion (35–39). These events precede entry of the viral nucleocapsid into the cytosol. Six-helix bundle formation and hemifusion appear to be co-dependent processes as lipids that promote positive spontaneous membrane curvature and block hemifusion also prevent completion of the trimer of hairpins fold (37).

How conserved sequences located outside the 6HB core domain of gp41, such as the N-terminally located polar segment adjacent to the fusion peptide, and the C-terminal Trp-rich MPR linking the C-helix to the TMD (40), contribute to the membrane fusion mechanism is not clearly understood. The MPR is of particular interest as it is a highly conserved region that overlaps with the C terminus of the Fuzeon inhibitor peptide and encompasses the epitopes of the two most broadly neutralizing mAbs available, 2F5 and 4E10 (41–43). The MPR is functionally relevant as simultaneous Ala replacements of the conserved Trp666-Trp670-Trp672-Trp678-Trp680 cluster block fusion pore opening (44), an effect that may be related to the lipid destabilizing properties of this sequence (45–48).

Like most sequences within the gp41 ectodomain, the MPR undergoes structural transitions in the fusion cascade. For example, the 2F5 epitope (Glu657-Trp670) is available for mAb binding in prefusion and prehairpin intermediate forms of Env but becomes occluded when the 6HB forms (21, 49–51). This change in antigenicity corresponds to a structural transition for the N-terminal portion (Glu657-Asp664) from an extended conformation to {alpha}-helix (30–34, 51). Helical conformation has also been observed for the MPR when bound to lipid (52). Antibody binding studies suggest that the polar segment and MPR occupy internal and external locations, respectively, in prefusion gp120-gp41 complexes (17, 20, 21). By contrast, these sequences would become apposed at the membrane-interactive end of the fusion-activated trimer of hairpins by formation of the 6HB. Consistent with this idea, extension of the 6HB core to include residues 528–535 of the polar segment and residues 669–677 of the MPR confers stability to a trimer of hairpins model protein (53).

In this study we assessed the functional role of the polar segment and MPR. Alanine replacement of conserved residues within the MPR did not affect cell-cell fusion but resulted in 8–120-fold reductions in viral entry. Simultaneous Ala replacement of MPR residues with Leu537 of the polar segment inhibited cell-cell fusion, viral entry, and sCD4-induced dissociation of gp120 from the Env complex. By using a trimer of hairpins model of gp41, we found that simultaneous mutations in the polar segment and MPR destabilized the membrane interactive end of this fusion-activated structure. We propose that the MPR has distinct roles in different stages of the fusion cascade.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
HIV-1 Env Expression Vectors—Preparation of the cytomegalovirus promoter-driven HIV-1AD8 Env expression vector, pCDNA3.1-AD8env, is described elsewhere (54). In vitro mutagenesis of the gp41 region was carried out by overlap extension PCR techniques. Bacteriophage T7 promoter-driven Env expression vectors based on pTM.1 (55) were generated by transferring the HIV-1AD8 env fragment bounded by NdeI and XhoI, from pCDNA3.1-AD8env to pTMenv.2, giving pTM-AD8env (56). DNA sequences were confirmed using the ABI BigDye terminator reagent. Env was either expressed in a Rev-dependent manner from the cytomegalovirus promoter of pCDNA3.1-AD8env, or in a Rev-independent manner from the bacteriophage T7 promoter in pTM-AD8env (55, 56). For Rev-independent expression, the cells were cotransfected with pTM-AD8env and pCAG-T7, the latter directing expression of bacteriophage T7 RNA polymerase from a cytomegalovirus immediate-early enhancer and chicken beta-actin promoter (57). Alternatively, the pTM-AD8env-transfected cells were infected with the recombinant vaccinia virus vTF7.3, which directs expression of bacteriophage T7 RNA polymerase (55).

Cells—293T, BHK21, HeLa, and U373MG-CCR5 cells were maintained in Dulbecco's modification of minimal essential medium, 10% fetal calf serum (complete medium) and transfected with expression vectors using FuGENE 6 (Roche). U87.CD4.CCR5 cells were maintained in Dulbecco's modified minimal essential medium, 15% fetal calf serum supplemented with puromycin (0.1 mg/ml) and G418 (0.3 mg/ml).

Antibodies—Immunoglobulin G number 14 (IgG 14) was purified from the plasma of a HIV-1 positive individual using protein A-Sepharose (Amersham Biosciences). Anti-gp41 mAbs were obtained through the AIDS Research and Reference Reagent Program, NIAID, National Institutes of Health, from G. Lewis (C8 (58)), S. Zolla-Pazner (126-6, 98-6 (59)), and R. Myers (Md.1 (60)). Goat anti-Env 2–3 (nonglycosylated HIV-1SF2 gp120) antibody was obtained through the AIDS Research and Reference Reagent Program, NIAID, National Institutes of Health, from K. Steimer (61).

Processing of Env Glycoproteins—At 24-h post-transfection, 293T cells were lysed for 10 min on ice in phosphate-buffered saline (PBS) containing 1% Triton X-100, 0.02% sodium azide, 1 mM EDTA. Lysates were clarified by centrifugation for 10 min at 10,000 x g at 4 °C prior to SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose and probed with mAb C8. The immunoblots were developed with horse-radish peroxidase-conjugated rabbit IgG to mouse Ig and enhanced chemiluminescence or with Alexa Fluor 680-conjugated goat anti-mouse Ig (Invitrogen) and scanning in a LI-COR Odyssey infrared imager.

Biosynthetic Labeling and Immunoprecipitation—293T cells were cotransfected with pTM-AD8env and pCAG-T7 plasmids. At 48-h post-transfection, the cells were incubated for 30 min in cysteine and methionine-deficient medium (MP Biomedicals, Seven Hills, New South Wales, Australia), and then labeled for either 20 or 45 min with 150 µCi of Tran35S-label (MP Biomedicals). The cells were then washed and chased in complete medium for 6 h prior to lysis. All incubations were performed at 37 °C in 5% CO2. Cell lysates and clarified culture supernatants were immunoprecipitated with various antibodies and protein G-Sepharose and subjected to SDS-PAGE under reducing conditions, as described previously (54). The labeled proteins were visualized by scanning in a Fuji phosphorimager.

Luciferase Reporter Assay of Cell to Cell Fusion—293T effector cells (250,000 cells per well of a 12-well culture plate) were cotransfected with pCDNA3.1-AD8env or pTM-AD8env and pCAG-T7 plasmids. BHK21 target cells (250,000 cells per well of a 12-well culture plate) were cotransfected with pT4luc (62) and pc.CCR5 (obtained from the AIDS Research and Reference Reagent Program from N. Landau (63)). At 24 h post-transfection, targets and effectors were each resuspended in 1 ml of complete medium; targets (100 µl) were co-cultured with effectors (100 µl) in a 96-well plate for a further 18 h at 37 °C. The cells were assayed for luciferase activity using the Promega SteadyGlo reagent (Promega, Madison, WI).

Lipid Mixing Assays—Lipid mixing assays were performed as described previously (64). HeLa effector cells (200,000 cells per well of a 6-well plate) were transfected with wild type or mutant Env-expression vectors. At 5 h post-transfection, the cells were infected with vTF7.3. At 18 h post-transfection, Env expressing cells were labeled with 2 µM DiO (3,3'-dioctadecyloxacarbocyanine perchlorate; green fluorescence, excitation at 484 nm, emission at 501 nm (Molecular Probes, Eugene, OR)), whereas target U373MG-CCR5 cells were labeled with 2 µM DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; red fluorescence, excitation at 549 nm, emission at 565 nm) for 2 min at room temperature. After washing twice with PBS, target cells were detached with PBS, 1 mM EDTA and added to an equivalent number of adherent effector cells. Following 2 h of co-culture at 37 °C, the cells were detached with trypsin, and fixed with 4% formaldehyde in PBS. Two-color fluorescence analysis was performed on an Epics XL flow cytometer. Cells displaying >4 and >70 arbitrary log units in the green and red wavelengths, respectively, were scored as double-fluorescent cells.

Single Cycle Infectivity Assays—Env-pseudotyped luciferase reporter viruses were produced by cotransfecting 293T cells (350,000 cells per well of a 6-well Linbro culture plate) with either 1 µg of pCDNA3.1-AD8env plus 1 µg of luciferase reporter virus vector, pNL4.3.Luc.R–E– (obtained from the AIDS Research and Reference Reagent Program from N. Landau (65)), or 1 µg of pTM-AD8env,1 µg of pCAG-T7 plus 1 µg of pNL4.3.Luc.R–E– using FuGENE 6. Supernatants containing pseudotyped virions were harvested at 72 h post-transfection and filtered through 0.45-µm filters. The infectivity of pseudotyped viruses was determined in U87.CD4.CCR5 cells (obtained from the AIDS Research and Reference Reagent Program from H. Deng and D. Littman (66)) as described (54). The protein composition of pseudotyped virions was assessed by first pelleting the virions from 9 ml of transfection supernatant through a 1.5-ml 25% (w/v) sucrose/PBS cushion (Beckman SW41 Ti rotor, 25,000 rpm, 2.5 h, 4 °C) followed by reducing SDS-PAGE in 7.5–15% polyacrylamide gradient gels and Western blotting with IgG 14 and goat anti-Env 2–3. The immunoblots were developed with IRDye 800CW-conjugated rabbit anti-human Ig (Rockland) or Alexa Fluor 680-conjugated donkey anti-goat Ig (Invitrogen), respectively, and scanning in a LI-COR Odyssey infrared imager.

Soluble CD4-induced Shedding of gp120—293T cells were transfected with Env expression plasmids as described above. At 24 h post-transfection, the cells were starved with methionine and cysteine-free medium for 30 min and then pulse labeled with 150 µCi of Tran35S-label for 45 min. The labeled cells were washed and then chased for 5 h with complete medium in the presence or absence of sCD4 (15 µg of sCD4 per 0.6 ml of medium). All incubations were at 37 °C in 5% CO2. The cells and culture supernatants were processed for immunoprecipitation with IgG 14 as described above.

Expression and Purification of MBP/gp41 Chimeras—The MBP/gp41(528-L-677) chimeras used in this study are derived from HIV-2ST and comprise the N-terminal polar segment and coiled coil (Ala528-Trp596), linked through Ser-Gly-Gly-Arg-Gly-Gly, to the C-terminal helix and membrane proximal segment (Trp610-Ser677) (32, 53). Residue numbering is according to the HXB2R convention. Ala528 of HIV-2ST gp41 was fused to the C terminus of MBP through an Asn-Ala linker incorporating a unique NotI site. Overlap extension PCR was used to generate the modified HIV-2ST gp41 ectodomain fragment: Ala528-Trp596-Ser-Gly-Gly-Arg: forward primer, 5'-ATAAGAATGCGGCCGCGATGGGCGCGGCGTCCTTGACG, reverse primer, 5'-ACCCCCACGACCCCCGGACCATGAATTTAGTTGCGCCTGGTC; Arg-Gly-Gly-Trp610-Ser677: forward primer, 5'-TCCGGGGGTCGTGGGGGTTGGGTAAATGACACCTTAACGCCTG, reverse primer, 5'-CTGAATATAGTCGACTTAGGAGGTTAAATCAAACCA. The PCR products were ligated into the MBP expression vector through NotI-SalI. The L537A, W666A, and L537A/W666A mutations were introduced to MBP/gp41(528-L-677) by overlap extension PCR. DNA sequences were confirmed using ABI prism BigDye terminator (Applied Biosystems, Foster City, CA). MBP/gp41 chimeras were induced in Escherichia coli strain BL21(DE3) and purified by amylose-agarose affinity chromatography (New England Biolabs) and gel filtration as described (53). MBP/gp41 trimers were proteolyzed with sequencing grade chymotrypsin (Roche) and analyzed by SDS-PAGE in 12–17% polyacrylamide gradient gels as described (53).


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 1.
Alignment of gp41 amino acid sequences. Secondary structures observed by NMR for the micelle-inserted fusion peptide and adjacent polar segment (24), are indicated by a line for random coil and cylinder for {alpha}-helix. The N- and C-terminal portions, respectively, of the coiled coil-forming N-helix and antiparallel C-helix of the 6-helix bundle core domain, as determined by x-ray crystallography (31, 33), are shown as truncated cylinders. The residues of the N-terminal polar segment and C-terminal MPR that were replaced with alanine in HIV-1AD8 and HIV-2ST gp41 in this study are shown and in bold type.

 
Mass Spectrometry—The digested proteins were mass analyzed by linear MALDI-MS (Bruker Daltonics, Germany) using 2,5-dihydroxybenzoic acid/5-methylsalicylic acid (9:1) (super DHB, Bruker Daltonics) as a matrix. The locations of chymotryptic cleavage sites were verified by in-gel tryptic digestion of the protein band in question. The excised bands were washed in 50 mM NH4HCO3/acetonitrile (1:1) (v/v) for 20 min and dried. Then, 0.3 µg of trypsin in 25 mM NH4HCO3 were added and the proteins digested for 2 h at 37°C. The resulting peptides were extracted in 5% formic acid/acetonitrile (1:1) (v/v) and dried. The extracts were dissolved in acetonitrile/water/formic acid (30:69:1) and applied to C18 ziptips. The peptides were eluted with DHB matrix in acetonitrile, 0.1% trifluoroacetic acid (aq) (6:4) and the eluates were analyzed by MALDI-MS in reflector mode.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Alanine Scan of the Fusion Peptide-proximal and Membraneproximal Regions of HIV-1AD8 gp41—Previously, we found that extension of the 6-helix bundle core of gp41 to include Ala528-Leu535, of the N-terminal polar segment, and Phe669-Ser677, within the MPR, conferred stability to a model of the HIV-2ST gp41 trimer of hairpins, MBP/gp41 (53). In this paper, we examined the functional relevance of these sequences following alanine replacement of component amino acids that are conserved in HIV-1, HIV-2, and SIV (Fig. 1). Met530 and Leu537 in the polar segment and Trp672 in the MPR are found in all HIV-1, HIV-2, and simian immunodeficiency virus isolates. Also within the MPR, Trp666, Phe673, and Ile675 are conserved in the majority of isolates. Notably, three conserved aromatic MPR residues contribute the majority of contacts with neutralizing mAb: Trp666 with 2F5 (51), Trp672 and Phe673 with 4E10 (67).

We first examined the synthesis of W666A, W672A, F673A, D674A, and I675A MPR mutants by Western blotting with mAb C8 (directed to the gp41 cytoplasmic domain) and found that the mutated gp160 precursors were expressed and processed to gp41 in a similar manner to wild type (Fig. 2A). The fusion activities of the MPR mutants were assessed using a luciferase reporter assay of cell-cell fusion. 293T effector cells were cotransfected with pCDNA3.1-AD8env and pCAG-T7, which directs expression of bacteriophage T7 RNA polymerase. In this assay, luciferase is induced after fusion of Env-293T effectors with CD4-CCR5-expressing BHK21 cell targets that contain the luciferase open reading frame under the control of the bacteriophage T7 promoter. Consistent with the observation that the mutants were expressed and cleaved normally, the MPR mutants were also able to mediate cell-cell fusion to a similar extent as wild type across a range of Env expression levels (Fig. 2B).

We next investigated whether luciferase reporter viruses pseudotyped with the MPR mutants could establish a single cycle of replication in U87.CD4.CCR5 target cells. Fig. 2C indicates that viral entry was reduced by ~120-fold for W666A and I675A, by 35-fold for W672A, and by 8-fold for F673A. By contrast, Ala replacement of the acidic side chain of Asp674 was tolerated (Fig. 2C). Western blotting of Env-pseudotyped HIV-1 particles that had been pelleted through 25% (w/v) sucrose indicated that the pseudovirions had retained gp120 in all cases (Fig. 2D). These data suggest that bulky hydrophobic side chains of the MPR are important for viral fusion but not cell-cell fusion.


Figure 2
View larger version (66K):
[in this window]
[in a new window]

  		FIGURE 2.
A, synthesis and processing of HIV-1AD8 Env glycoprotein mutants in 293T cells. 293T cells were transfected with the indicated amounts of wild type (WT) and mutated pCDNA3.1-AD8env expression plasmids. At 36 h post-transfection, the cells were lysed and subjected to reducing SDS-PAGE in 10% polyacrylamide gels. Proteins were transferred to nitrocellulose prior to Western blotting with mAb C8 and Alexa Fluor 680-conjugated goat immunoglobulins to mouse immunoglobulins and scanning in an LI-COR Odyssey infrared imager. Asterisk, ~80-kDa species corresponding in molecular mass to a biosynthetic intermediate or degradation product of gp160 (96). Representative of three independent experiments. B, cell-cell fusion activities of Env glycoprotein mutants using a luciferase reporter assay. 293T effector cells were cotransfected with the T7 polymerase expression vector, pCAG-T7, together with the indicated amounts of wild type (WT)or mutated pCDNA3.1-AD8env vectors. BHK21 target cells were cotransfected with pT4luc and pc.CCR5. At 18 h post-transfection, the effectors and targets were mixed and co-cultured for 24 h prior to lysis and assay for luciferase activity. Mean relative light units (RLU) ± standard error (error bars) is shown from at least 4 independent experiments. C, ability of Env mutants to mediate entry of Env-pseudotyped luciferase reporter viruses. Env-pseudotyped luciferase reporter viruses were prepared by cotransfecting 293T cells with 1 µg of pCDNA3.1-AD8env plus 1 µg of pNL4.3.Luc.R–.E–. At 72 h post-transfection, the virus-containing supernatants were filtered and used to infect U87.CD4.CCR5 monolayers for 18 h. The inoculum was then replaced with fresh medium and the cells assayed for luciferase activity at 52 h postinfection. Luciferase activity was normalized against the reverse transcriptase activity present in each virus inoculum. Mean RLU ± standard error from at least 3 independent experiments are shown. *, p < 0.05; **, p < 0.01 relative to wild type (two sample t test assuming unequal variances). D, protein content of virions. Env-pseudotyped virions were pelleted through a 25% sucrose cushion and subjected to SDS-PAGE under reducing conditions in 7.5–15% polyacrylamide gradient gels. Viral proteins were visualized by Western blotting with anti-Env 2–3 and Alexa Fluor 680-conjugated donkey anti-goat Ig (upper panel) or IgG 14 and IRDye 800CW-conjugated rabbit anti-human Ig (lower panel).

 
Effect of Simultaneous Mutations in the N-terminal Polar Segment and MPR—Our previous study with the MBP/gp41 chimeric model of the HIV-2 gp41 trimer of hairpins suggested that the polar segment and MPR confer trimer stability in a synergistic manner (53). Whereas these terminal sequences are likely to be brought together in late fusion forms of gp41 by 6-helix bundle formation, they appear to be spatially separate in the prefusion gp120-gp41 complex with the polar segment located in the interior of the trimer, whereas the MPR is external (17, 20, 21). We therefore examined the effects of simultaneous Ala substitutions in the polar segment and MPR at various stages of the fusion cascade. For these experiments, we used the bacteriophage T7 promoterdriven vector pTM.1 to overcome an Env expression defect resulting from disruption of stem-loop 2A of the Rev responsive element by the L537A mutation (data not shown). Western blotting of the L537A/W666A, L537A/W672A, L537A/F673A, L537A/D674A, and L537A/I675A double mutants, and L537A and W666A single mutants, revealed gp160 and gp41 at levels that were comparable with wild type (Fig. 3A). The gp120-anchoring abilities of gp41 L537A and the double mutants were confirmed by immunoprecipitation of pulsechase biosynthetically labeled Envtransfected 293T cells. Whereas similar amounts of gp160 were immunoprecipitated from the lysates of wild type and mutant Env-transfected cells, slightly more gp120 was shed into culture supernatants for the L537A-containing mutants (Fig. 3B), suggesting that L537A is associated with a subtle gp120-shedding phenotype. The M530A mutant was largely devoid of gp120, consistent with defective biosynthesis, and was not analyzed further.

The fusogenic abilities of L537A and L537A-containing double mutants were assessed in the luciferase reporter assay of cell-cell fusion. Fig. 3C shows that the L537A mutant was able to mediate fusion at wild type levels indicating that the shedding phenotype of this mutant observed in Fig. 3B was not associated with loss of cell-cell fusion function. However, the introduction of W666A, W672A, F673A, D674A, and I675A MPR mutations to the L537A background resulted in decreases in fusion activity of 80% for L537A/W666A, 69% for L537A/W672A, 61% for L537A/F673A, 56% for L537A/D674A, and 63% for L537A/I675A (Fig. 3C). The fusion activities of double mutants were significantly higher than the "No Env" control (p < 0.04). The M530A mutant lacked cell-cell fusion activity, consistent with its processing defect.


Figure 3
View larger version (66K):
[in this window]
[in a new window]

  		FIGURE 3.
Synthesis, processing, and cell-cell fusion activities of Env mutants expressed from the bacteriophage T7 promoter. A, 293T cells were cotransfected with pTM-Env and pCAG-T7 expression vectors. At 24 h post-transfection, the cells were lysed and subjected to reducing SDS-PAGE in 7.5–15% gradient polyacrylamide gels. The proteins were visualized by Western blotting with mAb C8 and Alexa Fluor 680-conjugated goat anti-mouse Ig. Representative of 3 independent experiments. B, gp120-anchoring ability of gp41 mutants. 293T cells were cotransfected with pTM-Env and pCAG-T7 vectors. At 48 h post-transfection, cells were labeled with Tran35S-label for 45 min and chased in complete medium for 6 h before lysis. Cell lysates (C) and clarified culture supernatants (S) were immunoprecipitated with IgG 14 and protein G-Sepharose. gp160 and gp120 bands were visualized following SDS-PAGE in 8–12% polyacrylamide gradient gels under reducing conditions and scanning in a PhosphorImager. The image was prepared from samples run on 2 gels in a single experiment; representative of three independent experiments. C, cell-cell fusion activity of gp41 mutants. 293T effector cells were cotransfected with pTM.1-Env and pCAG-T7 expression vectors. BHK21 target cells were cotransfected with pT4luc and pc.CCR5. At 24 h post-transfection, the effectors and targets were co-cultured for 18 h prior to assay for luciferase activity. Mean RLU ± standard error (error bars) from three independent experiments are shown. *, p < 0.03, relative to wild type (two sample t test assuming unequal variances). D, single cycle entry of Env-pseudotyped luciferase reporter viruses. Env-pseudotyped luciferase reporter viruses were prepared by cotransfecting 293T cells with 1 µg of pTM-Env, 1µg of pCAG-T7, and 1µg of pNL4.3.Luc.R–.E– and infectivity determined as described for Fig. 2D. {diamondsuit}, p < 0.005 relative to wild type; {dagger}, p < 0.004 relative to L537A (two sample t test assuming unequal variances). E, protein content of virions. Env-pseudotyped virions were pelleted through a 25% sucrose cushion and subjected to SDS-PAGE under reducing conditions in 7.5–15% polyacrylamide gradient gels. Viral proteins were visualized by Western blotting with anti-Env 2–3 and Alexa Fluor 680-conjugated donkey anti-goat Ig (upper panel) or IgG 14 and IRDye 800CW-conjugated rabbit anti human Ig (lower panel). F, conformation of the gp41 domain of the L537A/W666A mutant. Pulse-chase biosynthetically labeled wild type (w) and L537A/W666A-mutated (m) glycoproteins were immunoprecipitated with the indicated mAbs and visualized with a PhosphorImager following reducing SDS-PAGE on 8–12% gradient polyacrylamide gels. The image was prepared from samples run on 2 gels in a single experiment.

 
We next examined the ability of the combined L537A/MPR mutants to mediate viral entry. Wild type and L537A-containing mutant pseudovirions were produced for functional analysis by cotransfecting pTM-AD8env, pCAG-T7, and pNL4.3LucR–E– plasmids into 293T cells. The L537A mutation caused an ~10-fold decrease in viral entry competence, consistent with a lower level of gp120 retained on virions (Fig. 3, D and E). The introduction of W666A, W672A, F673A, and I675A MPR mutations to the L537A background ablated viral entry altogether even though the levels of virion-associated gp120 for the double mutants were similar to that observed for L537A. Of these double mutants, only L537A/D674A retained substantial entry activity, however, this function was decreased ~5-fold with respect to L537A (p < 0.0022). These data indicate that Leu537 in the polar segment is important for gp120-gp41 stability in both cellular and viral contexts. Furthermore, the data suggest that hydrophobic residues within the polar segment and MPR act together in membrane fusion in both cellular and viral contexts.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4.
Soluble CD4-induced shedding of gp120. 293T cells were transfected with pCDNA3.1-AD8env vectors (A) or cotransfected with pTM-Env plus pCAG-T7 vectors (B). At 24 h post-transfection, the cells were labeled with 150 µCi of Tran35S-label and then chased for 5 h in the absence (–) or presence (+) of 15 µg of sCD4. The culture supernatants (upper panels) and cell lysates (lower panels) were immunoprecipitated with IgG 14 and protein G-Sepharose and subjected to SDS-PAGE in 8–12% gradient gels followed by scanning in a PhosphorImager. The images were prepared from samples run on 4 gels in a single experiment.

 
The folding of the gp41 domain of a representative fusion-defective double mutant, L537A/W666A, was assessed by immunoprecipitation with the human mAbs, Md.1, 126.6, and 98.6, which bind to conformational epitopes in the C-terminal portion of the gp41 ectodomain (59, 60, 68). These mAbs are able to detect structural disturbances in gp41, as mutations in the gp41 coiled-coil sequence that block gp160 oligomerization also block epitope formation (68). Furthermore, mAbs 126.6 and 98.6 can recognize the hairpin form of gp41 (50). Monoclonal antibody C8 binds to a linear epitope in the cytoplasmic domain of gp41 (58) and was used as a positive control, whereas 12CA5 specific to the influenza hemagglutinin was included as an irrelevant antibody control. Fig. 3F shows that the L537A/W666A double mutant was efficiently immunoprecipitated by the mAbs indicating that the gp41 domain of this mutant acquires a global conformation that is similar to the wild type. sCD4-induced Shedding of gp120—Soluble CD4 triggers the formation of pre-hairpin intermediate conformations of gp41 (18, 19) and can activate Env-mediated fusion with target cells expressing only CCR5 or CXCR4 co-receptors (9). Functional gp120-gp41 complexes can also be induced to shed gp120 by sCD4 (69, 70). We examined whether sCD4 could induce gp120 shedding from cell surface-expressed [35S]Met/[35S]Cys-labeled gp120-gp41 to monitor the ability of the mutants to undergo conformational changes in response to receptor. The treatment of wild type and single MPR fusogenic mutants with sCD4 led to increased shedding of gp120 into culture supernatants when compared with untreated controls in all cases (Fig. 4A). These data indicate that gp120-gp41 complexes with single MPR mutations have similar stabilities to the wild type and that sCD4 can induce conformational changes in these mutants. Similar trends were observed for the fusogenic L537A and W666A mutants that had been expressed from the T7 promoter (Fig. 4B). However, relatively small or non-existent increases in sCD4-induced gp120 shedding were observed for the L537A/W666A, L537A/W672A, L537A/F673A, and L537A/I675A double mutants, which were defective for fusion and viral entry functions. The sCD4 shedding defect was not as apparent for L537A/D674A, consistent with the intermediate level of entry competence retained by this double mutant. These data suggest that gp120-gp41 complexes bearing L537A/W666A, L537A/W672A, L537A/F673A, and L537A/I675A double mutations cannot efficiently undergo CD4-induced conformational changes, perhaps contributing to their defective fusion and entry functions.

Effects of Mutations on Lipid Mixing—If fusion defective mutants lack the ability to undergo receptor-induced conformational changes in an efficient manner, then it follows that they will also be defective in mediating the lipid mixing stage of fusion. This stage is considered to precede pore expansion, which is measured in the luciferase assay. We tested this idea with a representative fusion defective mutant, L537A/W666A, by using flow cytometry to quantify lipophilic dye exchange between Env-expressing HeLa cells, labeled with the DiO green fluorescent probe, and U373MG-CCR5 targets labeled with the DiI red fluorescent probe (64). Fig. 5 indicates that Env glycoproteins with single L537A and W666A mutations retained wild type levels of lipid mixing activity, consistent with their cell-cell fusion abilities. The control X4 Env of HIV-1BH8 was unable to mediate lipid mixing with the U373MG-CCR5 targets, confirming the specificity of the assay. The L537A/W666A double mutation inhibited lipid mixing function by ~80%, indicating that these hydrophobic residues contribute jointly to the early stages of fusion.

Effects of Polar Linker and MPR Mutations on gp41 Trimer of Hairpins Stability—The fusion-activated trimer of hairpins/6HB conformation of gp41 is thought to facilitate virus-cell membrane fusion by bringing membrane-inserted fusion peptides and TMDs into close proximity. The polar and MPR segments link the 6HB to the terminal membrane-interactive sequences, and are therefore also likely to be juxtaposed in this late fusion form of gp41 (Fig. 6A). We asked whether these linking sequences play a role in the formation and/or stability of the trimer of hairpins. For these studies, we used an MBP/gp41 chimeric trimer of hairpins model, MBP/gp41(528-L-677), which comprises the HIV-2ST gp41 ectodomain fragment Ala528-Ser677 (53) with the interhelical disulfide-bonded region (Gly597-Pro609) replaced by Ser-Gly-Gly-Arg-Gly-Gly (32) (Fig. 6A). This modification enabled the purification of MBP/gp41 trimers by gel filtration (supplementary materials Fig. S1) at ~2 mg/liter of culture, corresponding to ~7-fold higher yields than versions containing the disulfide-bonded region (53). We found that chimeras comprised of corresponding HIV-1 sequences were either insoluble or presented as high-molecular weight aggregates that could not be analyzed further (data not shown). We used limited chymotrypsin proteolysis, which cleaves on the C-terminal side of Tyr, Phe, and Trp and to a lesser extent Leu, Met, Ala, Asp, and Glu, to examine how L537A, W666A, and L537A/W666A mutations affect the integrity of the MBP/gp41(528-L-677) trimer of hairpins.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 5.
Lipid mixing activities of Env mutants. HeLa effector cells were transfected with wild type (WT) or mutated Env expression vectors and at 5 h post-transfection, infected with the recombinant vaccinia virus vTF7.3. At 18 h post-transfection, effector and U373MG-CCR5 target cells were labeled with green (DiO) and red (DiI) fluorescent membrane probes, respectively, and co-cultured for 2 h at 37°C prior to detachment, fixation with 4% formaldehyde in PBS, and analysis of the green and red fluorescence in flow cytometry. The percentage lipid mixing activities were determined following the subtraction of background dye redistribution between empty vector-transfected/vTF7.3-infected HeLa cells labeled with DiO and U373MG-CCR5 targets labeled with DiI. The mean ± standard error (error bars) from six independent experiments are shown. *, p < 0.0001 relative to L537A and W666A (two sample t test assuming unequal variances).

 
Chymotrypsin released ~49, ~41, and <14.4-kDa products from wild type MBP/gp41(528-L-677) trimer (Fig. 6B, bands 1–3, respectively). When the protease:protein ratio was increased, an inverse correlation between the intensity of band 1 versus that of bands 2 and 3 was observed (Fig. 6, B–D); bands 2 and 3 are therefore chymotryptic peptides of band 1. Mass spectrometric analysis of the 1:40 and 1:5 chymotryptic digests of wild type MBP/gp41 indicated that the N- and C-helical sequences of the gp41 core remained largely intact with a number of their subfragments also released (Fig. 7). In-gel trypsin digestion and mass spectrometry was used to infer the C-terminal boundaries of bands 1 and 2. Trypsin cleaves on the C-terminal side of Lys and Arg; therefore peptides terminating with a residue preferred by chymotrypsin indicate the location of the chymotrypsin site(s). Trypsin treatment of band 1 released the chimeric peptide, MBP(Gln356-Thr367)-Asn-Ala-gp41(Ala528-Arg542) (2,760.4 Da), and the gp41 peptides, Thr543-Lys560 (1,994.2 Da) and Gln562-Lys585 (2,813.6 Da); we did not detect peptide(s) that were C-terminal to these sequences. Consistent with the lower molecular weight of band 2, MBP(Gln356-Thr367)-Asn-Ala-gp41(Ala528-Leu535) (2,016.9 Da) was identified as the C-terminal peptide. These data suggest that chymotrypsin cleaves initially on the C-terminal side of the N-helix to give band 1, followed by cleavage at Leu537 and/or Leu535 in the polar segment to give band 2; the diffuse band "3" is thus likely to comprise gp41 core domain peptides.

We compared the amounts of bands 1 and 2 released by chymotrypsin from wild type, L537A-, W666A-, and L537A/W666A-mutated MBP/gp41(528-L-677) to gauge how the mutations affected the stability of the polar segment (the polar segment contains the Leu535 and Leu537 chymotrypsin sites that give rise to band 2). The chymotrypsin sensitivity of W666A was almost identical to that of wild type, indicating that this mutation did not detectably affect the exposure of the preferred chymotrypsin site(s) (Fig. 6, B–D). Whereas treatment of the L537A mutant at a chymotrypsin:protein ratio of 1:40 released approximately wild type levels of band 1, the mutant resisted further cleavage to band 2 at higher protease:protein ratios (Figs. 6, B–D). This finding is consistent with removal of a favored chymotrypsin site (i.e. Leu537) and the alternate site, Leu535, remaining protected. Simultaneous L537A/W666A mutations led to increased amounts of band 2 being released at all protease:protein ratios when compared with the other constructs, and in particular with respect to L537A, which also lacks the Leu537 chymotrypsin site (Fig. 6, B–D). The N- and C-helical sequences of gp41 were detected by mass spectrometry at chymotrypsin:MBP/gp41 ratios of both 1:40 and 1:5 for L537A, W666A, and L537A/W666A-mutated MBP/gp41 suggesting that the structural integrity of the gp41 core is not substantially affected by mutations within the polar segment or MPR (Fig. 7). Thus, simultaneous L537A/W666A mutations increased the sensitivity of the polar segment to chymotrypsin cleavage at Leu535, suggesting that Leu537 and Trp666 cooperate in stabilizing the membrane-interactive end of the gp41 trimer of hairpins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that simultaneous Ala mutations in the polar segment and MPR inhibited Env fusogenicity and abolished viral entry competence. These functional defects correlated with reduced or absent sCD4-induced dissociation of gp120 from the gp120-gp41 complex and inhibition of the membrane fusion cascade prior to hemifusion. We also found that simultaneous L537A/W666A mutations, but not the individual L537A and W666A mutations, led to an increase in chymotrypsin sensitivity of the polar segment within the MBP/gp41(528-L-677) chimera, indicating that the stability of this region was affected by the double mutation in a trimer of hairpins structure. We infer a functional association between Leu537 in the polar segment and hydrophobic residues of the MPR in distinct stages of the fusion cascade: in the formation of a stable gp120-gp41 prefusion complex that is responsive to receptor engagement, and in stabilization of the membraneinteractive end of the late fusion trimer of the hairpins form of gp41.


Figure 6
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 6.
Effects of mutations on the chymotrypsin sensitivity of a trimer of hairpins model protein, MBP/gp41(528-L-677). A, schematic representation of MBP/gp41(528-L-677). Maltose-binding protein was linked through Asn-Ala to the HIV-2ST gp41 N-terminal sequence Ala528-Trp596, linked through Ser-Gly-Gly-Arg-Gly-Gly to the gp41 C-terminal sequence Trp610-Ser677. The upper sequence is that of HIV-1AD8, the lower sequence is that of HIV-2ST. The HXB2R numbering convention is used. The N-helix and C-helix are from the crystal structure of the HIV-1 gp41 6HB core (31). A monomer is depicted for clarity. B, chymotrypsin cleavage of wild type and mutated MBP/gp41(528-L-677). Purified MBP/gp41(528-L-677) trimers were treated with chymotrypsin for 10 min at 37 °C at the indicated protease:protein ratios and then subjected to SDS-PAGE under reducing conditions in 12–17% polyacrylamide gradient gels. The protein bands were visualized following staining with Coomassie Brilliant Blue and scanning in a LI-COR Odyssey infrared imager. C and D, quantitation of chymotryptic peptides. The intensities of bands 1 (C)and2(D) across the various protease:protein ratios were quantified using Odyssey version 1.2 software and expressed as a fraction of the corresponding mock treated protein. Representative results of three independent experiments is shown. WT, wild type.

 
Alanine replacement of individual MPR residues, Trp666, Trp672, Phe673, and Ile675, did not detectably affect cell-cell fusion, however, ~8–120-fold reductions in viral entry were observed. The fusion activities of the MPR mutants were indistinguishable over a range of Env concentrations, suggesting that small numbers of Env spikes and limited potential for cooperativity to overcome a minor functional defect is unlikely to account for the effects of the mutations on viral entry (22, 39, 71–75). The MPR has been shown to bind to and destabilize model membranes with cholesterol modulating this function (45–48, 76), therefore the functional differences observed for viral and cell surface Env forms may be attributed to the relative amounts of cholesterol in these membranes. The virion envelope is enriched in cholesterol and is less fluid than the plasma membrane with both attributes contributing to HIV-1 infectivity (77–80). The conformation and topology of the MPR in the lipidbound state has been suggested to be analogous to that of Trp-containing interfacial helical peptides, which destabilize membranes by introducing positive curvature strain to the outer leaflet of the bilayer (47, 48, 52). In the presence of cholesterol, such peptides penetrate the bilayer less deeply and exhibit decreased destabilizing activity than for membranes that lack cholesterol (81, 82). Viral entry defects are associated with Ala replacement of Trp666, Trp672, Phe673, and Ile675 (this study) as well as Leu669 and Leu679 (83), suggesting that a full complement of aromatic and hydrophobic residues is required for efficient MPR-mediated destabilization of the viral envelope. By contrast, at least 3 aromatic residues must be mutated simultaneously for cell-cell fusion function to be blocked (40).

The L537A polar segment mutant retained wild type levels of cell-cell fusion function despite having a subtle gp120 shedding phenotype for cellular Env, whereas a more pronounced loss of gp120 from the viral Env complex was linked to an ~10-fold decrease in viral entry competence. The L537A/MPR double mutants exhibited more severe cell-cell fusion defects and lacked viral entry function even though no further increase in gp120 shedding was observed. The results of mAb binding studies, together with the cryoelectron tomographic structure of the prefusion trimer solved by Zhu et al. (22) indicate that the gp41 ectodomain (excluding the MPR) forms the interior of the gp120-gp41 complex (17–20, 22). The L537A shedding phenotype may be due to the introduction of a cavity into this interior, which decreases the stability of the gp120-gp41 complex. The more pronounced shedding phenotype in virus suggests that the structure of the prefusion complex may be subtly altered following incorporation into virions, perhaps due to interactions with a more ordered cholesterol-enriched lipid bilayer and/or with the underlying matrix shell via the gp41 cytoplasmic tail (77, 84–86). The finding that a L49D mutation in matrix reduces virion-associated gp120 without affecting the levels of virion-associated gp41 indicates that gp41-matrix interactions can affect gp120-gp41 complex stability (87).


Figure 7
View larger version (54K):
[in this window]
[in a new window]

  		FIGURE 7.
Chymotryptic peptides of wild type and L537A-, W666A- and L537A/W66A-mutated MBP/gp41(528-L-677). Wild type (WT) and mutated MBP/gp41(528-L-677) derived from HIV-2ST was digested with chymotrypsin at protease:protein ratios (w/w) of 1:40 and 1:5 at 37 °C for 10 min. The digested proteins were mass analyzed by linear MALDI-MS using 2,5-dihydroxybenzoic acid:5-methylsalicylic acid (9:1) as a matrix. The identity of gp41 peptides were inferred from the observed molecular masses. Black, N-helix; gray, C-helix; dashed line, interhelical region.

 
The simultaneous removal of hydrophobic side chains from the polar segment and MPR in L537A/W666A, L537A/W672A, L537A/F673A, and L537A/I675A mutants led to marked decreases in cell-cell fusion and the abolition of viral entry. The spontaneous gp120 shedding phenotypes of the double mutants were similar to that of L537A for both cellular and viral forms of Env. Therefore, the more severe fusion and entry defects of the double mutants relative to the single mutants could not be simply attributed to further loss of gp120 from the Env complex. However, these functional defects correlated with decreased responsiveness of the mutant gp120-gp41 complexes to sCD4, suggesting that the mutants are activated less efficiently by receptor than is wild type. This idea is consistent with the observation that L537A/W666A (but not individual L537A and W666A mutants) was inhibited prior to the hemifusion stage of the fusion cascade. Leu537 therefore appears to act together with the MPR in the formation of a fusion competent gp120-gp41 complex. Lorizate and co-workers (88, 89) put forward a model whereby the N-terminal fusion peptide and MPR form a complex within prefusion Env, acting as a kinetic trap to halt fusion. This idea is based on the findings with synthetic peptides that fusion peptide-MPR interactions enhance mAb 2F5 binding (mAb 2F5 binds to the prefusion Env complex), and inhibit the membrane destabilizing properties of the component sequences. The synthetic fusion peptide-MPR complex is largely non-helical with a predominance of beta-turns, consistent with the structure of the Glu657-Asp664 sequence when bound to 2F5 (51). The fusion peptide-MPR prefusion clasp model theoretically brings the polar segment into proximity with the MPR. The loss of hydrophobicity in these 2 sequences due to L537A/MPR mutations may affect the fusion peptide-MPR clasp and its release following gp120-receptor interactions.

Previously, we reported that N- and C-terminal extension of the 6-helix bundle core to include the polar segment residues, Ala528-Leu535, and MPR residues, Phe669-Ser677, respectively, conferred thermal stability to a MBP/gp41 trimer of hairpins model protein (53). In the current study, we found that the L537A/W666A double mutation increased chymotrypsin sensitivity of the polar segment. The L537A mutation alone inhibited proteolysis of the polar segment, indicating that the upstream Leu535 site (Ile535 in AD8) is not readily accessed by chymotrypsin. By contrast, mutating L537A together with W666A led to enhanced proteolysis at Leu535. Proteases at limiting concentrations preferentially cleave protein substrates in mobile loops rather than within folded domains, suggesting that L537A/W666A disrupts the structural order at the end of the hairpin, enabling the protease to access Leu535. These observations indicate a structural link between Leu537 and Trp666 in stabilizing the membrane proximal end of the hairpin. However, because the individual L537A and W666A mutations did not increase the protease sensitivity of the polar segment, they are either unlikely to interact directly, or the effects of the mutations are mitigated by other contacts in this region.


Figure 8
View larger version (48K):
[in this window]
[in a new window]

  		FIGURE 8.
Model of the HIV-1 fusion cascade. The metastable state of the gp120-gp41 complex is released by binding of gp120 to CD4 and chemokine receptor (CKR). The majority of gp41 is occluded by the gp120 trimer. The MPR, which encompasses the mAb 2F5 and 4E10 epitopes, is exposed and adjacent to the viral envelope (A). Formation of the prehairpin intermediate of gp41 (B) precedes refolding of the core into a 6-helix bundle and hemifusion (C and D). Coil to helix transition in the polar segment creates a hydrophobic binding site for the MPR (E) completing the trimer of hairpins fold (F). Fusion peptide, orange cylinder; polar segment, purple tube (random coil)/purple cylinder ({alpha}-helix); coiled coil, pink cylinder; C-helix, light blue tube and cylinder; MPR, dark blue cylinder; TMD, hatched oblong. The cytoplasmic domain is not shown. Enlarged inset, theoretical model of the 6-helix bundle core (33) with an N-terminal helical extension (purple ribbon) to Ser528 at the fusion peptide-polar segment boundary (94). The extension creates a hydrophobic binding site (Met530, Ile535, Leu537) for the MPR (the 2F5- and 4E10-bound structures are shown (51, 67)). The model was prepared with Swiss PDB Viewer (version 3.7) and POV Ray (version 3.5.1).

 
A potential mechanism whereby these terminal sequences could confer stability to gp41 is illustrated by HA2 of influenza virus. In this case, low pH activates a loop-to-helix transition that extends the N terminus of the central coiled coil by some 100 Å that is terminated by an N-cap. The surface grooves of the coiled coil terminus close to the N-cap act as a hydrophobic docking site for the C-terminal MPR (90). Mutations designed to destabilize this terminal interaction have been reported to block hemifusion and/or pore opening, depending on the mutation and assay system used (91, 92). Random coil and {alpha}-helical conformations have been observed for the polar segment in synthetic peptide models of the N-terminal portion of the gp41 prehairpin intermediate (24, 26, 93) with propagation of helical structure beyond the core to Ser528 at the fusion peptide-polar segment boundary improving the thermal stability of the gp41 core domain (94). Helical extension of the coiled coil N terminus indicates that Met530, Ile535, and Leu537 form the grooves of the coiled coil (Met530 and Leu537 occupy g positions of the hydrophobic heptad repeat, whereas Ile535 occupies e) potentially creating a hydrophobic docking site for the MPR when brought into proximity by 6HB formation. Such interactions would "zip up" the membrane-interactive end of the gp41 hairpin, aiding in the apposition of virus and target cell membranes and contributing additional free energy to membrane fusion. The idea that the MPR acquires structural order in the context of the trimer of hairpins is supported by our earlier analysis of a MBP/gp41 chimera comprising the entire gp41 ectodomain where the Phe669-Ser677 MPR sequence resisted chymotrypsin despite the presence of 7 potential protease sites (53).

These scenarios are illustrated in Fig. 8, which summarizes the HIV-1 fusion cascade. The mAb 2F5 and 4E10 epitopes are available for antibody binding in prefusion and prehairpin intermediate forms of gp41 (Fig. 8, A and B, respectively) but are lost or occluded by 6-helix bundle formation (21, 49–51) (Fig. 8, D–F). According to the model put forward by Lorizate et al. (88, 89), the fusion peptide and MPR interact to form a prefusion clasp that is released by gp120-receptor interactions (Fig. 8A). Membrane-inserted fusion peptides are depicted as {alpha}-helices with the adjacent polar segment as a random coil, based on NMR and Fourier transform infrared spectroscopy findings (24, 25, 93, 95) (Fig. 8, B–D). However, it should be noted that beta-strand conformations have also been documented for this sequence (26). Formation of the 6-helix bundle core leads to hemifusion (37) (Fig. 8D), and a coil-to-helix transition in the polar segment (94) (Fig. 8, D and E) creates a hydrophobic binding site (Met530, Ile535, and Leu537) for the MPR (Fig. 8E, inset), completing the trimer of hairpins fold (Fig. 8F). A possible mechanism for mAb 2F5 neutralization, which binds to the MPR in the prefusion Env structure, is the blockade of terminal interactions between the polar segment and MPR that are required to complete the trimer of hairpins fold. In summary, our data indicate that the N-terminal polar segment and C-terminal MPR of gp41 are functionally linked in distinct phases of the fusion cascade, acting synergistically to form a fusion-competent prefusion gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.


   FOOTNOTES
 
* This work was supported by the National Health and Medical Research Council of Australia Grants 296200 and 345413, American Foundation for AIDS Research Grant 106610-36-RGNT, and Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 1 Both authors contributed equally to this work. Back

2 Present address: Program Infection and Cancer, Abt. F010 and INSERM U701, Deutsches 10 Krebsforschungszentrum, Heidelberg, Germany. Back

3 To whom correspondence should be addressed: 85 Commercial Rd., Melbourne, Victoria 3004, Australia. Fax: 613-9282-2100; E-mail: apoumbourios{at}burnet.edu.au.

4 The abbreviations used are: Env, envelope glycoprotein; HIV-1, human immunodeficiency virus type 1; N-helix, N-terminal coiled coil forming {alpha}-helix of gp41; C-helix, C-terminal {alpha}-helix of gp41; MPR, membrane proximal region; TMD, transmembrane domain; mAb, monoclonal antibody; sCD4, soluble CD4; DiO, 3,3'-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; MBP, maltose-binding protein; MALDI, matrixassisted laser desorption ionization; BHK, baby hamster kidney; gp, glycoprotein; 6HB, 6-helix bundle; SIV, simian immunodeficiency virus. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Basmaciogullari, S., Babcock, G. J., Van Ryk, D., Wojtowicz, W., and Sodroski, J. (2002) J. Virol. 76, 10791–10800[Abstract/Free Full Text]
  2. Chen, B., Vogan, E. M., Gong, H., Skehel, J. J., Wiley, D. C., and Harrison, S. C. (2005) Nature 433, 834–841[CrossRef][Medline] [Order article via Infotrieve]
  3. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W. A. (1998) Nature 393, 648–659[CrossRef][Medline] [Order article via Infotrieve]
  4. Rizzuto, C. D., Wyatt, R., Hernandez-Ramos, N., Sun, Y., Kwong, P. D., Hendrickson, W. A., and Sodroski, J. (1998) Science 280, 1949–1953[Abstract/Free Full Text]
  5. Hoffman, N. G., Seillier-Moiseiwitsch, F., Ahn, J., Walker, J. M., and Swanstrom, R. (2002) J. Virol. 76, 3852–3864[Abstract/Free Full Text]
  6. Pastore, C., Nedellec, R., Ramos, A., Pontow, S., Ratner, L., and Mosier, D. E. (2006) J. Virol. 80, 750–758[Abstract/Free Full Text]
  7. Rosen, O., Sharon, M., Quadt-Akabayov, S. R., and Anglister, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13950–13955[Abstract/Free Full Text]
  8. Pierson, T. C., and Doms, R. W. (2003) Curr. Top. Microbiol. Immunol. 281, 1–27[Medline] [Order article via Infotrieve]
  9. Salzwedel, K., Smith, E. D., Dey, B., and Berger, E. A. (2000) J. Virol. 74, 326–333[Abstract/Free Full Text]
  10. Colman, P. M., and Lawrence, M. C. (2003) Nat. Rev. Mol. Cell. Biol. 4, 309–319[CrossRef][Medline] [Order article via Infotrieve]
  11. Eckert, D. M., and Kim, P. S. (2001) Annu. Rev. Biochem. 70, 777–810[CrossRef][Medline] [Order article via Infotrieve]
  12. Gallaher, W. R., Ball, J. M., Garry, R. F., Griffin, M. C., and Montelaro, R. C. (1989) AIDS Res. Hum. Retroviruses 5, 431–440[Medline] [Order article via Infotrieve]
  13. Kielian, M., and Rey, F. A. (2006) Nat. Rev. Microbiol. 4, 67–76[CrossRef][Medline] [Order article via Infotrieve]
  14. Lamb, R. A., Paterson, R. G., and Jardetzky, T. S. (2006) Virology 344, 30–37[CrossRef][Medline] [Order article via Infotrieve]
  15. Skehel, J. J., and Wiley, D. C. (2000) Annu. Rev. Biochem. 69, 531–569[CrossRef][Medline] [Order article via Infotrieve]
  16. Xu, Y., Lou, Z., Liu, Y., Pang, H., Tien, P., Gao, G. F., and Rao, Z. (2004) J. Biol. Chem. 279, 49414–49419[Abstract/Free Full Text]
  17. Finnegan, C. M., Berg, W., Lewis, G. K., and DeVico, A. L. (2002) J. Virol. 76, 12123–12134[Abstract/Free Full Text]
  18. Furuta, R. A., Wild, C. T., Weng, Y., and Weiss, C. D. (1998) Nat. Struct. Biol. 5, 276–279[CrossRef][Medline] [Order article via Infotrieve]
  19. He, Y., Vassell, R., Zaitseva, M., Nguyen, N., Yang, Z., Weng, Y., and Weiss, C. D. (2003) J. Virol. 77, 1666–1671[Abstract/Free Full Text]
  20. Sattentau, Q. J., Zolla-Pazner, S., and Poignard, P. (1995) Virology 206, 713–717[CrossRef][Medline] [Order article via Infotrieve]
  21. Dimitrov, A. S., Jacobs, A., Finnegan, C. M., Stiegler, G., Katinger, H., and Blumenthal, R. (2007) Biochemistry 46, 1398–1401[CrossRef][Medline] [Order article via Infotrieve]
  22. Zhu, P., Liu, J., Bess, J., Jr., Chertova, E., Lifson, J. D., Grise, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847–852[CrossRef][Medline] [Order article via Infotrieve]
  23. Zanetti, G., Briggs, J. A., Grunewald, K., Sattentau, Q. J., and Fuller, S. D. (2006) PLoS Pathog. 2, e83[CrossRef][Medline] [Order article via Infotrieve]
  24. Chang, D. K., Cheng, S. F., and Trivedi, V. D. (1999) J. Biol. Chem. 274, 5299–5309[Abstract/Free Full Text]
  25. Gordon, L. M., Mobley, P. W., Pilpa, R., Sherman, M. A., and Waring, A. J. (2002) Biochim. Biophys. Acta 1559, 96–120[Medline] [Order article via Infotrieve]
  26. Sackett, K., and Shai, Y. (2005) J. Mol. Biol. 350, 790–805[CrossRef][Medline] [Order article via Infotrieve]
  27. Root, M. J., Kay, M. S., and Kim, P. S. (2001) Science 291, 884–888[CrossRef][Medline] [Order article via Infotrieve]
  28. Wild, C., Oas, T., McDanal, C., Bolognesi, D., and Matthews, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10537–10541[Abstract/Free Full Text]
  29. Wild, C. T., Shugars, D. C., Greenwell, T. K., McDanal, C. B., and Matthews, T. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9770–9774[Abstract/Free Full Text]
  30. Caffrey, M., Cai, M., Kaufman, J., Stahl, S. J., Wingfield, P. T., Covell, D. G., Gronenborn, A. M., and Clore, G. M. (1998) EMBO J. 17, 4572–4584[CrossRef][Medline] [Order article via Infotrieve]
  31. Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997) Cell 89, 263–273[CrossRef][Medline] [Order article via Infotrieve]
  32. Tan, K., Liu, J., Wang, J., Shen, S., and Lu, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12303–12308[Abstract/Free Full Text]
  33. Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., and Wiley, D. C. (1997) Nature 387, 426–430[CrossRef][Medline] [Order article via Infotrieve]
  34. Yang, Z. N., Mueser, T. C., Kaufman, J., Stahl, S. J., Wingfield, P. T., and Hyde, C. C. (1999) J. Struct. Biol. 126, 131–144[CrossRef][Medline] [Order article via Infotrieve]
  35. Blumenthal, R., Sarkar, D. P., Durell, S., Howard, D. E., and Morris, S. J. (1996) J. Cell Biol. 135, 63–71[Abstract/Free Full Text]
  36. Markosyan, R. M., Cohen, F. S., and Melikyan, G. B. (2005) Mol. Biol. Cell 16, 5502–5513[Abstract/Free Full Text]
  37. Melikyan, G. B., Markosyan, R. M., Hemmati, H., Delmedico, M. K., Lambert, D. M., and Cohen, F. S. (2000) J. Cell Biol. 151, 413–423[Abstract/Free Full Text]
  38. Melikyan, G. B., Barnard, R. J., Abrahamyan, L. G., Mothes, W., and Young, J. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 8728–8733[Abstract/Free Full Text]
  39. Muñoz-Barroso, I., Durell, S., Sakaguchi, K., Appella, E., and Blumenthal, R. (1998) J. Cell Biol. 140, 315–323[Abstract/Free Full Text]
  40. Salzwedel, K., West, J. T., and Hunter, E. (1999) J. Virol. 73, 2469–2480[Abstract/Free Full Text]
  41. Binley, J. M., Wrin, T., Korber, B., Zwick, M. B., Wang, M., Chappey, C., Stiegler, G., Kunert, R., Zolla-Pazner, S., Katinger, H., Petropoulos, C. J., and Burton, D. R. (2004) J. Virol. 78, 13232–13252[Abstract/Free Full Text]
  42. Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Ruker, F., and Katinger, H. (1993) J. Virol. 67, 6642–6647[Abstract/Free Full Text]
  43. Zwick, M. B., Labrijn, A. F., Wang, M., Spenlehauer, C., Saphire, E. O., Binley, J. M., Moore, J. P., Stiegler, G., Katinger, H., Burton, D. R., and Parren, P. W. (2001) J. Virol. 75, 10892–10905[Abstract/Free Full Text]
  44. Munoz-Barroso, I., Salzwedel, K., Hunter, E., and Blumenthal, R. (1999) J. Virol. 73, 6089–6092[Abstract/Free Full Text]
  45. Saez-Cirion, A., Nir, S., Lorizate, M., Agirre, A., Cruz, A., Perez-Gil, J., and Nieva, J. L. (2002) J. Biol. Chem. 277, 21776–21785[Abstract/Free Full Text]
  46. Shnaper, S., Sackett, K., Gallo, S. A., Blumenthal, R., and Shai, Y. (2004) J. Biol. Chem. 279, 18526–18534[Abstract/Free Full Text]
  47. Suarez, T., Nir, S., Goni, F. M., Saez-Cirion, A., and Nieva, J. L. (2000) FEBS Lett. 477, 145–149[CrossRef][Medline] [Order article via Infotrieve]
  48. Suarez, T., Gallaher, W. R., Agirre, A., Goni, F. M., and Nieva, J. L. (2000) J. Virol. 74, 8038–8047[Abstract/Free Full Text]
  49. de Rosny, E., Vassell, R., Jiang, S., Kunert, R., and Weiss, C. D. (2004) J. Virol. 78, 2627–2631[Abstract/Free Full Text]
  50. Gorny, M. K., and Zolla-Pazner, S. (2000) J. Virol. 74, 6186–6192[Abstract/Free Full Text]
  51. Ofek, G., Tang, M., Sambor, A., Katinger, H., Mascola, J. R., Wyatt, R., and Kwong, P. D. (2004) J. Virol. 78, 10724–10737[Abstract/Free Full Text]
  52. Schibli, D. J., Montelaro, R. C., and Vogel, H. J. (2001) Biochemistry 40, 9570–9578[CrossRef][Medline] [Order article via Infotrieve]
  53. Lay, C. S., Wilson, K. A., Kobe, B., Kemp, B. E., Drummer, H. E., and Poumbourios, P. (2004) FEBS Lett. 567, 183–188[CrossRef][Medline] [Order article via Infotrieve]
  54. Poumbourios, P., Maerz, A. L., and Drummer, H. E. (2003) J. Biol. Chem. 278, 42149–42160[Abstract/Free Full Text]
  55. Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A., and Fuerst, T. R. (1990) Nature 348, 91–92[CrossRef][Medline] [Order article via Infotrieve]
  56. Poumbourios, P., Ahmar, W. E., McPhee, D. A., and Kemp, B. E. (1995) J. Virol. 69, 1209–1218[Abstract]
  57. Takikawa, S., Ishii, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y., and Miyamura, T. (2000) J. Virol. 74, 5066–5074[Abstract/Free Full Text]
  58. Abacioglu, Y. H., Fouts, T. R., Laman, J. D., Claassen, E., Pincus, S. H., Moore, J. P., Roby, C. A., Kamin-Lewis, R., and Lewis, G. K. (1994) AIDS Res. Hum. Retroviruses 10, 371–381[Medline] [Order article via Infotrieve]
  59. Xu, J.-Y., Gorny, M. K., Palker, T., Karwowska, S., and Zolla-Pazner, S. (1991) J. Virol. 65, 4832–4838[Abstract/Free Full Text]
  60. Myers, R., Meiller, T., Falkler, W., Jr., Patel, J., and Joseph, J. (1993) in Abstracts of the 93rd General Meeting of the American Society for Microbiology, American Society for Microbiology, Washington, D. C.
  61. Haigwood, N. L., Shuster, J. R., Moore, G. K., Lee, H., Skiles, P. V., Higgins, K. W., Barr, P. J., George-Nascimento, C., and Steimer, K. S. (1990) AIDS Res. Hum. Retroviruses 6, 855–869[Medline] [Order article via Infotrieve]
  62. Maerz, A. L., Drummer, H. E., Wilson, K. A., and Poumbourios, P. (2001) J. Virol. 75, 6635–6644[Abstract/Free Full Text]
  63. 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., and Landau, N. R. (1996) Nature 381, 661–666[CrossRef][Medline] [Order article via Infotrieve]
  64. Baär, S., and Alizon, M. (2004) J. Virol. 78, 811–820[Abstract/Free Full Text]
  65. Connor, R. I., Chen, B. K., Choe, S., and Landau, N. R. (1995) Virology 206, 935–944[CrossRef][Medline] [Order article via Infotrieve]
  66. Björndal, A., Deng, H., Jansson, M., Fiore, J. R., Colognesi, C., Karlsson, A., Albert, J., Scarlatti, G., Littman, D. R., and Fenyo, E. M. (1997) J. Virol. 71, 7478–7487[Abstract]
  67. Cardoso, R. M., Zwick, M. B., Stanfield, R. L., Kunert, R., Binley, J. M., Katinger, H., Burton, D. R., and Wilson, I. A. (2005) Immunity 22, 163–173[CrossRef][Medline] [Order article via Infotrieve]
  68. Poumbourios, P., Wilson, K. A., Center, R. J., El Ahmar, W., and Kemp, B. E. (1997) J. Virol. 71, 2041–2049[Abstract]
  69. Sattentau, Q. J., Moore, J. P., Vignaux, F., Traincard, F., and Poignard, P. (1993) J. Virol. 67, 7383–7393[Abstract/Free Full Text]
  70. Willey, R. L., Martin, M. A., and Peden, K. W. (1994) J. Virol. 68, 1029–1039[Abstract/Free Full Text]
  71. Chertova, E., Bess, J. W., Jr., Crise, B. J., Sowder, I. R., Schaden, T. M., Hilburn, J. M., Hoxie, J. A., Benveniste, R. E., Lifson, J. D., Henderson, L. E., and Arthur, L. O. (2002) J. Virol. 76, 5315–5325[Abstract/Free Full Text]
  72. Kuhmann, S. E., Platt, E. J., Kozak, S. L., and Kabat, D. (2000) J. Virol. 74, 7005–7015[Abstract/Free Full Text]
  73. Yang, X., Kurteva, S., Ren, X., Lee, S., and Sodroski, J. (2005) J. Virol. 79, 12132–12147[Abstract/Free Full Text]
  74. Yang, X., Kurteva, S., Ren, X., Lee, S., and Sodroski, J. (2006) J. Virol. 80, 4388–4395[Abstract/Free Full Text]
  75. Zhu, P., Chertova, E., Bess, J., Jr., Lifson, J. D., Arthur, L. O., Liu, J., Taylor, K. A., and Roux, K. H. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15812–15817[Abstract/Free Full Text]
  76. Lorizate, M., Cruz, A., Huarte, N., Kunert, R., Perez-Gil, J., and Nieva, J. L. (2006) J. Biol. Chem. 281, 39598–39606[Abstract/Free Full Text]
  77. Aloia, R. C., Tian, H., and Jensen, F. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5181–5185[Abstract/Free Full Text]
  78. Campbell, S. M., Crowe, S. M., and Mak, J. (2002) AIDS 16, 2253–2261[CrossRef][Medline] [Order article via Infotrieve]
  79. Guyader, M., Kiyokawa, E., Abrami, L., Turelli, P., and Trono, D. (2002) J. Virol. 76, 10356–10364[Abstract/Free Full Text]
  80. Liao, Z., Graham, D. R., and Hildreth, J. E. (2003) AIDS Res. Hum. Retroviruses 19, 675–687[CrossRef][Medline] [Order article via Infotrieve]
  81. Egashira, M., Gorbenko, G., Tanaka, M., Saito, H., Molotkovsky, J., Nakano, M., and Handa, T. (2002) Biochemistry 41, 4165–4172[CrossRef][Medline] [Order article via Infotrieve]
  82. Matsuzaki, K., Sugishita, K., Ishibe, N., Ueha, M., Nakata, S., Miyajima, K., and Epand, R. M. (1998) Biochemistry 37, 11856–11863[CrossRef][Medline] [Order article via Infotrieve]
  83. Zwick, M. B., Jensen, R., Church, S., Wang, M., Stiegler, G., Kunert, R., Katinger, H., Burton, D. R., Parker, C. E., Deterding, L. J., Hager-Braun, C., Binley, J. M., Schulke, N., Moore, J. P., and Tomer, K. B. (2005) J. Virol. 79, 1252–1261[Abstract/Free Full Text]
  84. Bhattacharya, J., Repik, A., and Clapham, P. R. (2006) J. Virol. 80, 5292–5300[Abstract/Free Full Text]
  85. Murakami, T., and Freed, E. O. (2000) J. Virol. 74, 3548–3554[Abstract/Free Full Text]
  86. Wyma, D. J., Kotov, A., and Aiken, C. (2000) J. Virol. 74, 9381–9387[Abstract/Free Full Text]
  87. Davis, M. R., Jiang, J., Zhou, J., Freed, E. O., and Aiken, C. (2006) J. Virol. 80, 2405–2417[Abstract/Free Full Text]
  88. Lorizate, M., Gomara, M. J., de la Torre, B. G., Andreu, D., and Nieva, J. L. (2006) J. Mol. Biol. 360, 45–55[CrossRef][Medline] [Order article via Infotrieve]
  89. Lorizate, M., de la Arada, I., Huarte, N., Sanchez-Martinez, S., de la Torre, B. G., Andreu, D., Arrondo, J. L., Nieva, J. L., Katinger, H., Kunert, R., Cruz, A., Perez-Gil, J., Gomara, M. J., Mingarro, I., Perez-Paya, E., Hermann, K., Basanez, G., Saez-Cirion, A., Iloro, I., Melikyan, G., Nir, S., and Agirre, A. (2006) Biochemistry 45, 14337–14346[CrossRef][Medline] [Order article via Infotrieve]
  90. Chen, J., Skehel, J. J., and Wiley, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8967–8972[Abstract/Free Full Text]
  91. Borrego-Diaz, E., Peeples, M. E., Markosyan, R. M., Melikyan, G. B., and Cohen, F. S. (2003) Virology 316, 234–244[CrossRef][Medline] [Order article via Infotrieve]
  92. Park, H. E., Gruenke, J. A., and White, J. M. (2003) Nat. Struct. Biol. 10, 1048–1053[CrossRef][Medline] [Order article via Infotrieve]
  93. Jaroniec, C. P., Kaufman, J. D., Stahl, S. J., Viard, M., Blumenthal, R., Wingfield, P. T., and Bax, A. (2005) Biochemistry 44, 16167–16180[CrossRef][Medline] [Order article via Infotrieve]
  94. Sackett, K., Wexler-Cohen, Y., and Shai, Y. (2006) J. Biol. Chem. 281, 21755–21762[Abstract/Free Full Text]
  95. Chang, D. K., Cheng, S. F., and Chien, W. J. (1997) J. Virol. 71, 6593–6602[Abstract]
  96. Earl, P. L., Moss, B., and Doms, R. W. (1991) J. Virol. 65, 2047–2055[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Champagne, A. Shishido, and M. J. Root
Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil
J. Biol. Chem., February 6, 2009; 284(6): 3619 - 3627.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. S.-L. Chen, P. Yang, P.-Y. Ke, H.-F. Li, W.-E. Chan, D.-K. Chang, C.-K. Chuang, Y. Tsai, and S.-C. Huang
Identification of the LWYIK Motif Located in the Human Immunodeficiency Virus Type 1 Transmembrane gp41 Protein as a Distinct Determinant for Viral Infection
J. Virol., January 15, 2009; 83(2): 870 - 883.
[Abstract] [Full Text] [PDF]

Home page
 Microbiol. Mol. Biol. Rev.Home page
M. Montero, N. E. van Houten, X. Wang, and J. K. Scott
The Membrane-Proximal External Region of the Human Immunodeficiency Virus Type 1 Envelope: Dominant Site of Antibody Neutralization and Target for Vaccine Design
Microbiol. Mol. Biol. Rev., March 1, 2008; 72(1): 54 - 84.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/32/23104    most recent
M703485200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bellamy-McIntyre, A. K.
Right arrow Articles by Poumbourios, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bellamy-McIntyre, A. K.
Right arrow Articles by Poumbourios, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement