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Journal of Virology, October 2005, p. 12231-12241, Vol. 79, No. 19
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.19.12231-12241.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Fusion by a Membrane-Interactive Domain in the gp41 Cytoplasmic Tail

Stéphanie Wyss,1 Antony S. Dimitrov,3 Frédéric Baribaud,2 Terri G. Edwards,2 Robert Blumenthal,3 and James A. Hoxie1*

Department of Medicine, Hematology-Oncology Division,1 Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,2 Center for Cancer Research Nanobiology Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 217023

Received 8 April 2005/ Accepted 5 July 2005


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ABSTRACT
 
Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.


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INTRODUCTION
 
Human immunodeficiency virus (HIV) entry is mediated by highly coordinated interactions between HIV envelope glycoproteins (Env), gp120 with CD4 and a chemokine receptor (mainly CCR5 or CXCR4), and gp41 with the target cell membrane. This process involves extensive conformational changes in gp120 initiated by the binding of gp120 to CD4, which leads to a structural rearrangement in gp41 and insertion of its amino terminus into the host cell membrane, with subsequent lipid mixing of viral and cell membranes (7, 24, 67). CD4-independent variants of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) have also been described that can interact directly with chemokine receptors and enter cells without a need for CD4 triggering (25-27, 31, 46, 49, 68, 72). For at least some of these viruses, mutations in gp120 expose epitopes that are characteristically induced after CD4 binding, some of which include a highly conserved chemokine receptor binding domain on the gp120 core (38, 47).

gp120 and gp41 are produced from a gp160 precursor glycoprotein that is cleaved by a cellular protease and organized on the virion surface as spikes of heterotrimers. gp120 contains binding sites for CD4 and chemokine receptors, while gp41 contains an amino-terminal fusion domain and two heptad repeat regions (HR1 and HR2) in its ectodomain, a single membrane-spanning domain, and a long cytoplasmic tail (CT) of approximately 150 amino acids. Although there are numerous examples of changes in gp120 and the gp41 ectodomain that contribute to cell tropism, cytopathogenicity, fusion kinetics, and neutralization sensitivity, the gp41 CT can also exert significant effects on Env function. For HIV and SIV, point mutations, and particularly truncations, can increase fusogenicity (2, 33, 35, 61, 80, 81, 88, 100), Env surface expression (50, 100), and the incorporation of Env into virions (15, 55, 97, 100) as well as alter the biochemical and immunologic properties of the Env ectodomain (28, 29, 81). There are also numerous examples among other retroviruses in which mutations in the cytoplasmic tail can impact Env function (12, 13, 20, 41, 70, 74, 75, 84, 99).

The HIV and SIV cytoplasmic tails contain a number of functional domains, including (i) a Yxx{Phi} motif that mediates binding to AP2 µ chains (8, 10, 11, 64), clathrin-dependent endocytosis (8, 11, 64, 77, 79), and basolateral sorting of Env in polarized cells (53, 66); (ii) one or more palmitoylated cysteines implicated in targeting Env to lipid rafts (9, 76); (iii) three highly conserved alpha-helical "lentivirus lytic peptide" domains (LLP-1, LLP-2, and LLP-3) implicated in interacting with the plasma membrane, decreasing bilayer stability, altering membrane ionic permeability, and mediating cell killing (18, 19, 21, 30, 43, 45, 58, 59, 87); (iv) calmodulin binding domains (40, 60, 82, 85); and (v) a sorting motif at the C terminus that can alter the intracellular localization of Env (92). Poorly defined regions of gp41 have also been implicated in interacting with viral matrix proteins during virion assembly (15, 32, 33, 54, 91). Recent reports have indicated that this interaction also modulates Env function in that gp41 is more stably associated with immature than with mature viral particles (91) and that cleavage of the p55 Gag precursor protein by the viral protease is required to generate Envs with maximal fusogenicity (62, 90). This last finding has led to the view that in immature virions the association of distal elements of the CT with unprocessed Gag proteins may serve to limit Env fusogenicity until Gag is cleaved as virions mature and are released from the cell (62, 90).

Interestingly, for a CD4-independent variant of HIV-1 termed 8x that utilizes CXCR4 in the absence of CD4 (38, 49), a frameshift mutation (fs) in the CT resulting in a prematurely truncated tail of 27 amino acids is required for its CD4 independence and contributes to both the exposure of CD4-induced epitopes and an increased neutralization sensitivity (28, 29, 49). Subsequent studies demonstrated that it was the premature truncation and not the frameshift per se that was responsible for these effects (29). However, the CT domain(s) responsible and the mechanisms by which a tail truncation affects the structure and function of the Env ectodomain are unknown.

In this report, we identify a region of the gp41 CT responsible for altering the conformation of the CD4-independent HIV-1 8x Env ectodomain and more precisely define its effect on Env function. We show that truncations of gp41 proximal to the first palmitoylated cysteine (at position 764) are sufficient to increase the fusion efficiency of the parental HXBc2 Env and to expose CD4-induced epitopes on gp120. Moreover, using a highly quantitative assay to measure the kinetics of Env fusion, we show that these mutations, as well as truncation at the beginning of the flanking LLP-2 domain (at position 771), lead to a two- to fourfold increase in the rate of fusion compared to that of wild-type Envs. These results were observed with both CXCR4- and CCR5-expressing target cells and could not be explained by differences in the levels of Env expression on the cell surface. These results suggest that distal to the membrane-spanning domain a subsequent interaction of the most proximal alpha-helical region (LLP2) with cellular membranes serves to constrain Env function. Thus, on both virions where Env is likely associated with Gag proteins and cells in the absence of Gag, the Env cytoplasmic tail reduces Env fusogenicity and may provide a mechanism through which the membrane-disruptive potential of the Env ectodomain is restrained during intracellular transport and virion assembly.


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MATERIALS AND METHODS
 
Viral envelopes and mutagenesis. The parental Env clones for HXB, 8x, JRFL, and 89.6 were expressed from pSP73 as previously described (38). Frameshift mutations were introduced into the cytoplasmic tail at amino acid 706 (numbering for the HXB sequence) using a Quickchange site-directed mutagenesis kit (Stratagene) as described previously (29). Stop codons were introduced into the HXB cytoplasmic tail at positions 733, 753, 764, 771, and 808 using the primers 5'-GACAGGCCCGAAGGATAAGAAGAAGAAGGTGGAGAG-3' and 5'-CTCTCCACCTTCTTCTTCTTATCCTTCGGGCCTGTC-3' for position 733, 5'-GATTAGTGAACGGATCCTAGGCACTTATCTGGGACG-3' and 5'-CGTCCCAGATAAGTGCCTAGGATCGTTCACTAATC-3' for position 753, 5'-CTGCGGAGCCTGTGACTCTTCAGCTACC-3' and 5'-GGTAGCTGAAGAGTCACAGGCTCCGCAG-3' for position 764, 5'-CAGCTACCACCGCTAGAGAGACTTACTCTTG-3' and 5'-CAAGAGTAAGTCTCTCTAGCGGTGGTAGCTG-3' for position 771, and 5'-GGAGTCAGGAACTATAGAATAGTGCTGTTAGCTTGC-3' and 5'-GCAAGCTAACAGCACTATTCTATAGTTCCTGACTCC-3' for position 808. An additional construct, with a stop codon at position 771 and a Cys-to-Ala mutation at position 764, was made using the primers 5'-CTGCGGAGCCTGGCCCTCTTCAGCTAC-3' and 5'-GTAGCTGAAGAGGGCCAGGCTCCGCAG-3'. Stop codons were introduced into JRFL and 89.6 at position 748 (Fig. 1) with the primers 5'-GAGACAGATCCGGATGATTAGTGAACGG-3' and 5'-CCGTTCACTAATCATCCGGATCTGTCTC-3' for JRFL and 5'-CAGATCCGGTCCATAAGTGAACGGATTCTTG-3' and 5'-CAAGAATCCGTTCACTTATGGACCGGATCTG-3' for 89.6.



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  		FIG. 1. Mutagenesis of the HIV-1 gp41 cytoplasmic domain. Sequence alignments of gp41 cytoplasmic tails are shown for HXBc2, JRFL, and 89.6, using the numbering for HXBc2. Highlighted domains include the C-terminal region of the membrane-spanning domain (msd) and the amphipathic alpha-helical domains LLP-1, LLP-2, and LLP-3 (18, 43, 45, 58, 59, 87). A largely conserved palmitoylated Cys is shown (solid arrow) prior to the beginning of LLP-2, as is the position of a frameshift mutation (gray arrow) described for the CD4-independent variant of HXBc2, 8x (49). Mutations introduced into HXBc2 are shown, including stop codons (asterisks) at the indicated positions, i.e., amino acids 733 (Ile), 753 (Leu), 764 (Cys), 771 (Leu), and 808 (Lys), and the 8x fs mutation at position 706. An additional mutant (HXB-C764A/771*) is shown containing a stop codon and a Cys-to-Ala mutation at position 764 and a stop codon at position 771. For JRFL and 89.6, the mutations introduced included the 8x fs mutation and stop codons introduced to produce tails comparable in length to those with the fs mutations.

Cells. HeLa cells expressing CD4, CCR5, and/or CXCR4 have been previously described (36), as have the human T-cell lines SupT1 (52) and CEM-CCR5 (provided by the NIH AIDS Reagent Repository). QT6 and HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (DMEM-10), and SupT1 and CEM CCR5 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. All culture media contained 100 units/ml penicillin G and 100 µg/ml streptomycin sulfate.

Antibodies. The human monoclonal antibody 17b was obtained from James Robinson (Tulane University Medical School, New Orleans, LA) (86), and a human fluorescein isothiocyanate (FITC)-conjugated anti-gp120 monoclonal antibody used to assess Env surface expression on HeLa cells was obtained from ImmunoDiagnostic, Inc.

Cell-cell fusion assays. A modification of a previously described cell-cell fusion assay (78) was used (52). Briefly, effector Env-expressing QT6 cells were infected with vaccinia virus strain WR and transfected by the standard calcium phosphate method with Env plasmids and pSP64.vE/E.T7 RNAP, a plasmid expressing T7 RNA polymerase under the regulation of the synthetic vaccinia virus early and late promoter (16, 52). Target quail QT6 cells were transfected with CXCR4 or CCR5 and CD4 expression plasmids and the luciferase reporter gene under the control of the T7 promoter. On the next day, effector cells were mixed with target cells, and cell-cell fusion was assessed 6 h later. Fusion was measured by the quantification of luciferase activity in cell lysates after the addition of luciferase substrate (Promega) with a Wallac 1450 Microbeta Plus luminometer. For inhibition experiments, various concentrations of enfuvirtide (ENF; Trimeris, Durham, NC) were added to the effector cells at the time of mixing with the target cells, and the inhibition of fusion was measured as the percent reduction in luciferase activity. The standard error of the mean was calculated from the values obtained in each experiment (i.e., three experiments done in triplicate).

Evaluation of CD4-induced epitopes. The binding assay used to assess the expression of CD4-induced epitopes on gp120 was previously described in detail (28). Cells were infected with recombinant vaccinia virus vTF1.1 expressing T7 polymerase, transfected by Ca3(PO4)2 with a plasmid containing the Env protein of interest, and then incubated overnight at 37°C. On the next day, Env-bearing cells were washed once with phosphate-buffered saline (PBS). Cells were resuspended in binding buffer (50 mM HEPES [pH 7.4], 5 mM magnesium chloride, 1 mM calcium chloride, 0.5% bovine serum albumin) and incubated with 1 µg of monoclonal antibody/106 cells for 20 min at room temperature. Cells were washed once with PBS and resuspended in 50 µl of binding buffer. Iodinated anti-human immunoglobulin G (100,000 cpm in 50 µl of binding buffer) was added to the cells, and the mixture was incubated for 1 h at room temperature. Cells were collected on Brandel-grade GF/B filters with wash buffer (same as the binding buffer, but with 150 mM sodium chloride and without bovine serum albumin) by using a cell harvester. Counts on filters were determined by using a Wallac Wizard 1470 automatic gamma counter. The percent binding was determined by dividing the counts on the filters by the input radioactivity. Background binding was measured with pcDNA3-transfected cells and subtracted from the sample signals obtained. The standard error of the mean was calculated from the values obtained in each experiment (i.e., three experiments done in triplicate).

Evaluation of Env surface expression. The surface expression of Env on HeLa cells was quantified by fluorescence image cytometry as previously described (23). HeLa cells were infected with recombinant vaccinia virus vTF7-3 expressing T7 polymerase and transfected with pSP73 expressing Envs as described above. Transfected cells were placed on ice and washed twice with ice-cold washing buffer (phosphate-buffered saline with calcium and magnesium [Quality Biological, Inc.] containing 0.1% bovine serum albumin and 0.02% sodium azide). Nonspecific antibody binding was blocked by incubating cells with 2% mouse serum in D-PBS for 30 min on ice. Cells were then washed twice and incubated (1 h on ice) in 2% mouse serum supplemented with 1 µg/ml of the FITC-conjugated anti-gp120 monoclonal antibody. Cells were washed and covered with 1 ml of washing buffer per well prior to microscopic observation. Images of FITC-stained cells were collected, and the fluorescence intensity was quantified for each image pixel. By using the built-in histogram function of MetaMorph 4.0 (Universal Imaging Co., Downingtown, PA) software, the numbers of pixels having the same (but higher than zero) fluorescence intensities were collected, and the data were transferred to a Microsoft Excel data sheet. HeLa cells infected with vaccinia virus vTF7-3 or with the empty plasmid were used as controls for nonspecific staining.

Kinetic assays for cell fusion. The kinetics of cell fusion were quantified by a dye transfer assay as described previously (36). Wild-type and mutant Envs were expressed in HeLa cells by transfection with the respective env-expressing plasmids. Each plasmid was added to a final concentration of 0.02 to 0.06 mg/ml Lipofectamine solution in DMEM-0 (without serum) in a glass tube, vortexed for 1 min, and then equilibrated for 1 h at room temperature. Aliquots (100 µl) of each plasmid suspension were added to HeLa cells that had previously been plated in 12-well plates (2 x 105 cells per well) and were incubated for 4 h at 37°C, with shaking every 10 min. For the last incubation hour, vaccinia virus vTF7-3 was added at a multiplicity of infection of 10. After 4 h, 1 ml DMEM with 10% serum was added per well, and cells were incubated overnight at 31°C. Transfected HeLa cells were easily distinguishable from target SupT1 or CEM-CCR5 cells by their morphology when observed by microscopy with differential interference contrast. At the start of an incubation, target cells preloaded with calcein AM (Molecular Probes, Inc., Eugene, OR) were added on top of the plated transfected HeLa cells and incubated at 25°C for 25 min. The cell mixture was then washed once with medium to remove the unbound target cells, covered with medium prewarmed at 37°C, and incubated for 20 min, 45 min, 80 min, or 2 h at 37°C in 5% CO2. Reactions were terminated at each time point by removing the medium from the wells and covering the cells with ice-cold RPMI without serum. Fusion was determined as the ratio between Env-expressing cells stained with calcein and the total number of Env-expressing cells in contact target cells counted from microscopic images. Images were collected, and fused cells were counted using MetaMorph 4.0 software (Universal Imaging Co., Downingtown, PA) (42). Statistical differences in fusion kinetics between pairs of data were calculated using a paired t test with a cutoff P value of <0.05 for statistical significance. All images were collected using a Nikon 200TE inverted microscope supplied with a PlanFluor 20x, ELWD, 0.45-numerical-aperture objective using single beam-splitter cubes (Nikon B-2E/C) for calcein and FITC staining.


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RESULTS
 
Construction of gp41 cytoplasmic tail mutants. A frameshift (fs) mutation in the CT of 8x, a CD4-independent variant of HXBc2, contributes to fusion on CXCR4 in the absence of CD4, exposes CD4-induced epitopes on gp120, and increases neutralization sensitivity (28, 29, 49). This effect results from the premature termination of gp41 rather than nonsense mutations caused by the fs, since similar effects were seen when a stop codon was introduced into HXBc2 at position 733 to produce a comparably sized CT of only 27 amino acids (29). To map the determinants for this effect, a panel of mutants was constructed with stop codons introduced based on a model of gp41 in which the CT contains at least the following four membrane-interactive regions (43, 94): a largely conserved palmitoylated Cys at position 764 (9, 76, 94) and three amphipathic helical regions located at positions 828 to 856, 770 to 795, and 789 to 815 (termed LLP-1, LLP-2, and LLP-3, respectively) that have been implicated to interact with the plasma membrane (18, 19, 21, 30, 43, 45, 58, 59) and to bind to calmodulin (40, 60, 82, 85) (Fig. 1). A stop codon at Ile-733 (733*) generated a tail of 27 amino acids, identical in length to the 8x CT, while stop codons at Leu-753 (753*), Cys-764 (764*), Leu-771 (771*), and Lys-808 (808*) generated truncated tails of 47, 58, 65, and 102 amino acids, respectively. These truncations were located prior to membrane-interactive domains (733* and 753*), at the first palmitoylation site (C764*), at the beginning of LLP-2 (L771*), and within LLP-3, between LLP-2 and LLP-1 (808*) (Fig. 1). A construct was also generated that contained a stop codon at position 771 in combination with an alanine substitution at Cys-764, which ablated the palmitoylation site (C764A/771*). For some experiments, mutations analogous to 8x-fs and Ile-733* were introduced into the R5-tropic HIV-1 JRFL Env and the dual-tropic 89.6 Env (see Materials and Methods). The fs mutations in JRFL (JRFL-fs) and 89.6 (89.6-fs) generated CTs of 42 amino acids. Stop codons were also introduced into JRFL and 89.6 (at position 748) to generate CTs identical in length to Envs with the fs mutation (JRFL-stop and 89.6-stop, respectively) (Fig. 1).

Effects of cytoplasmic tail truncations on fusion efficiency. The constructs shown in Fig. 1 were first evaluated with a cell-cell fusion assay that has been used extensively to characterize Env function, CD4 independence, and chemokine receptor utilization (31, 49, 51, 78). For this assay, Envs are expressed in QT6 effector cells, and fusion is assessed with QT6 target cells expressing CD4 and a chemokine receptor, either CXCR4 or CCR5. As reported previously (28), 8x exhibited a two- to threefold increase in fusion activity relative to HXBc2 (Fig. 2). These differences could not be attributed to differences in Env surface expression (28, 29, 49). A similar increase in fusion activity was seen using HXBc2 constructs containing either the fs (HXB-fs) or 733* mutation. Although the 753* and 764* constructs showed a similar enhancement of fusion, the 771* and 808* mutants exhibited fusion levels comparable to that of HXBc2. Thus, truncations of the cytoplasmic tail up to and including the palmitoylated Cys proximal to LLP-2 increased fusogenicity.



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  		FIG. 2. Increased fusogenicity with truncations in the gp41 cytoplasmic tail. The fusion activity of HIV-1 Envs was assessed in cell-cell fusion assays with target cells expressing CD4 and CXCR4, as described in Materials and Methods. Envs included those of parental HXBc2 (HXB), 8x (a CD4-independent variant of HXBc2), and HXB-fs (HXBc2 containing a frameshift mutation at position 706 in the cytoplasmic tail) and HXBc2-derived Envs containing stop codons in their cytoplasmic tails at the indicated amino acid positions (see Fig. 1). The results of luciferase activity measurements were normalized to the level of fusion obtained with parental HXB. Envs with cytoplasmic tails terminating at amino acid 764 or before exhibited greater fusion activities, whereas Envs with stop codons at positions 771 and 808 had activities similar to that of HXB. The data were compiled from three experiments, with each done in triplicate. Standard errors of the means are shown.

Effects of cytoplasmic tail truncations on exposure of CD4-induced epitopes. The 8x CT fs, as well as the 733* mutation, increases the exposure of CD4-induced epitopes recognized by the anti-gp120 monoclonal antibodies 17b and 48d (28), which overlap a conserved gp120 core domain involved in CCR5 and CXCR4 binding (5, 48, 89). This effect was seen for both R5- and X4-tropic Envs (29). To identify the domains responsible for this effect, 17b epitope exposure was assessed with the panel of HXBc2 constructs shown in Fig. 1. QT6 cells were transfected with the indicated constructs and evaluated for 17b binding using a radioimmunobinding assay (28). As seen previously, 8x, HXB-fs, and the 733* mutant each exhibited an approximately threefold increase in 17b reactivity relative to wild-type HXBc2 (Fig. 3). Although similar results were seen with the 753* and 764* mutants, the 771* and 808* mutants exhibited low levels of 17b exposure comparable to that of parental HXBc2. Thus, similar to fusion activity, truncations proximal to the start of LLP-2 increased the exposure of a CD4-induced epitope.



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  		FIG. 3. Exposure of CD4-induced epitopes after gp41 cytoplasmic tail truncations. The binding of antibody 17b, which reacts with a CD4-induced epitope on gp120, was assessed on Envs expressed in QT6 cells by using a previously described binding assay (28, 29). The results were normalized to the amount of binding seen on the CD4-independent 8x Env, which has been shown to expose this epitope in the absence of CD4 binding (38, 49). Results are shown for the panel of HXB-derived Envs evaluated in Fig. 1. The data are from three experiments, with each done in triplicate. Standard errors of the means are shown.

Effects of cytoplasmic tail truncations on HXBc2 fusion kinetics. The effects of CT truncations on fusion kinetics were next determined using a dye transfer assay (36). Envs were expressed on HeLa cells and evaluated over time for the ability to fuse with SupT1 cells, which express both CD4 and CXCR4. In previous studies, 8x exhibited an approximately threefold increase in the rate of fusion compared to that of its CD4-dependent parental Env from HXBc2 (36). As shown in Fig. 4 and Table 1, an HXBc2 Env with a full-length cytoplasmic tail and one with a stop codon at position 808 exhibited half-maximal fusion times of 65.8 ± 8 min and 82.4 ± 15 min, respectively, which were comparable to those previously determined for HIV-1/IIIB, from which the HXBc2 clone was derived (36). In contrast, the 753*, 764*, and 771* mutants exhibited markedly increased fusion kinetics, with half-maximal fusion times of 27.4 ± 3, 29.7 ± 2, and 32.1 ± 1 min, respectively. Fusion of the C764A/L771* mutant was also accelerated, with a half-maximal fusion time of 23.2 ± 1 min. P values for the differences in half-maximal fusion kinetics compared to HXBc2 were 0.0015, 0.0016, 0.0019, and 0.0008 for the 753*, 764*, 771*, and C764A/L771* mutants, respectively. Therefore, all Envs truncated at the beginning of or prior to LLP-2 exhibited increased fusion kinetics compared to a full-length tail or one with a stop codon between LLP-2 and LLP-1. Notably, although the 771* construct did not exhibit enhanced fusogenicity or exposure of CD4 epitopes, it did show enhanced fusion kinetics in this assay.



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  		FIG. 4. Fusion kinetics of HXBc2-derived Envs with gp41 cytoplasmic tail truncations on SupT1 cells. The panel of HXBc2-derived Envs shown in Fig. 1 were expressed in HeLa cells, and the rate of fusion was assessed on SupT1 cells (CD4+ and CXCR4+) labeled with calcein AM (see Materials and Methods). An additional Env is shown containing a stop codon at position 771 in combination with a Cys-to-Ala mutation at position 764, which ablated a palmitoylation site (Fig. 1). Lines represent fits to the sigmoidal equation f = a/{1 – exp[–b(t – t1/2)]} (36). Values for half-maximal fusion times are shown in Table 1. Envs with cytoplasmic tails truncated at or before position 771 exhibited accelerated fusion kinetics compared to Env 808* and wild-type HXBc2.


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  		TABLE 1. Summary of fusion kinetics of HXBc2, 89.6, and JRFL Envs with and without truncation mutations in their cytoplasmic tailsa

Effects of cytoplasmic tail truncations on fusion kinetics of R5- and R5/X4-tropic Envs. We next determined if the effects of CT truncations on fusion kinetics also occurred for R5 (JRFL)- and dual (89.6)-tropic Envs. As shown in Fig. 1, constructs were created that contained the fs mutation present in 8x and stop codons that generated cytoplasmic tails of 42 amino acids, which were equal in length to these Envs with the fs mutation.

For 89.6-based Envs, the fusion kinetics of 89.6-stop and 89.6-fs on CEM/CCR5 cells were markedly accelerated compared to that of the wild type, with half-maximal fusion occurring at 33.6 ± 3 min and 43.1 ± 3 min, respectively, compared to 111.8 ± 64 min for parental 89.6 (Table 1; Fig. 5A). The P values for the differences between the mutants and parental 89.6 were 0.007 for 89.6-stop and 0.007 for 89.6-fs. Similar results were seen with JRFL-stop and JRFL-fs, which exhibited half-maximal fusion times of 23.6 ± 1 and 45.7 ± 4 min, respectively, compared to 120.3 ± 72 min for parental JRFL, with P values of 0.08 for JRFL-stop and 0.147 for JRFL-fs. The higher P values for the JRFL mutants were likely the result of the large experimental error for the wild-type Env. Nonetheless, these results for CEM/CCR5 cells were quite similar to those with 89.6. Interestingly, on SupT1 cells, which express CXCR4 but lack CCR5, wild-type 89.6 was considerably faster (37.8 ± 3 min) than on CEM/CCR5 cells (118.8 ± 64 min). The fusion kinetics on SupT1 cells for 89.6-stop and 89.6-fs were also somewhat increased relative to that of parental 89.6, with half-maximal fusion times of 27.3 ± 2 and 28.5 ± 1 min, respectively (Table 1; Fig. 5B), although this difference was of borderline statistical significance (P values of 0.08 for 753* and 0.11 for JRFL-fs). Nonetheless, although the cell type could affect the fusion kinetics of both X4- and R5-tropic Envs, overall Envs with truncated CTs fused with faster kinetics than those with long CTs, and for HXBc2 this increase was seen with mutations occurring at the beginning of or proximal to the start of LLP-2.



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  		FIG. 5. Fusion kinetics of R5- and dual-tropic Envs with prematurely truncated cytoplasmic tails. (A) Fusion kinetics of JRFL and 89.6 Envs with a CT frameshift mutation at a position analogous to that in 8x or a premature stop codon at position 748 were assessed on CEM/CCR5 cells as described in Materials and Methods. Times to half-maximal fusion are shown in Table 1. (B) The dual-tropic 89.6 Envs were also evaluated with the CD4+ CXCR4+ cell line SupT1. Although the differences were less impressive, 89.6-fs and -stop Envs each exhibited faster kinetics than the 89.6 wild type (Table 1).

Surface expression of Envs with cytoplasmic tail truncations. We next determined if differences in fusion kinetics could have resulted from differences in the cell surface expression of Env. HeLa cells transfected with the Envs shown in Fig. 1 were stained with an anti-gp120 monoclonal antibody, and surface expression was determined using quantitative fluorescence microscopy (see Materials and Methods). No differences were observed between any of the constructs, including those lacking the palmitoylation site at Cys-746, which has been implicating in directing Env to lipid rafts on the cell surface (Fig. 6) (9, 76). These findings were similar to our previous studies using flow cytometry, which found no differences in surface expression between HXBc2, 8x, and HXB-fs Envs (28, 29, 49). Thus, differences in Env surface expression could not account for the differences in Env fusion efficiency, the exposure of CD4-induced epitopes, or the fusion kinetics noted above.



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  		FIG. 6. Surface expression of Env constructs. HXBc2-derived Envs containing a stop codon at position 733 or the 8x frameshift mutation were expressed in HeLa cells, and surface expression was evaluated 24 h later by fluorescence imaging microscopy (see Materials and Methods). Controls included parental HXBc2 and the CD4-independent 8x Env. The negative controls were HeLa cells infected with vTF or transfected with a PSP plasmid. No differences in Env expression were seen for these or the other plasmid shown in Fig. 1.

Effects of cytoplasmic tail mutations on sensitivity to enfuvirtide. We previously reported that compared to its parental Env from HXBc2, the 8x Env exhibits decreased sensitivity to the fusion entry inhibitor ENF, a finding that was attributed to the faster kinetics of this CD4-independent Env on CXCR4-expressing target cells (36, 39). When similar assays were performed with HXBc2-derived Envs with truncated CTs, namely, the HXB-fs and 733* mutants, both were less sensitive to ENF than HXBc2, with 50% inhibitory concentrations (IC50s) of 0.108 and 0.269 µg/ml for HXB-fs and 733*, respectively, compared to 0.035 µg/ml for wild-type HXBc2 (Fig. 7). When JRFL and JRFL-fs Envs were compared on CCR5-expressing target cells, similar results were seen, with an IC50 of 0.108 µg/ml for JRFL-fs compared to one of >0.5 µg/ml for wild-type JRFL (not shown). Therefore, for both JRFL and HXBc2, accelerated fusion kinetics caused by CT truncations correlated on CCR5- and CXCR4-expressing target cells with a reduced sensitivity to ENF.



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  		FIG. 7. Sensitivity of cytoplasmic tail mutant Envs to enfuvirtide inhibition. The sensitivity of fusion to inhibition by enfuvirtide is shown for the HXBc2-derived Envs HXB-fs and 733* compared to the parental HXBc2 and CD4-independent 8x Envs. For each Env, the % fusion on SupT1 cells in the absence of inhibitor is shown, and data were plotted using Sigma Plot. IC50s are shown for each Env.

Effects of restoring a full-length cytoplasmic tail to CD4-independent 8x Env. The 8x Env is able to fuse using CXCR4 in the absence of CD4, a finding attributed both to gp120 mutations that expose domains involved in CXCR4 binding and to poorly understood effects of the CT truncation induced by the fs mutation noted above (28, 29, 49). To assess more fully the effects of the fs mutation on coreceptor utilization, we performed fusion assays with QT6 cells that expressed CXCR4 with or without CD4. The Envs utilized were those from HXBc2, 8x, HXB-fs, and 8x in which the fs mutation was corrected to restore a full-length CT (8x/HXB-TM).

Consistent with our previous results (38, 49) HXBc2 fusion was restricted to target cells that coexpressed CD4 and CXCR4, while CD4-independent fusion for 8x occurred at approximately half the level observed in the presence of CD4 (Fig. 8). Although HXB-fs showed increased fusion, as noted above (Fig. 2), this Env remained strictly CD4 dependent, indicating that neither its increased fusion kinetics nor the exposure of CD4-induced epitopes (Fig. 3) (28) was sufficient for CD4-independent fusion. Interestingly, when a full-length cytoplasmic tail was restored in 8x, this Env not only exhibited reduced levels of fusion but also became largely CD4 dependent (Fig. 8). Thus, in addition to the other effects noted above, the presence or absence of a full-length CT had a marked functional effect on the CD4 dependence of coreceptor utilization.



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  		FIG. 8. Effects of cytoplasmic tail on CD4 independence. Cell-cell fusion assays were performed with QT6 cells transfected with control pCDNA3 or with CD4, CXCR4, or CD4 in combination with CXCR4. The Envs used were those of HXB, 8x, and HXB-fs and an 8x Env containing a full-length cytoplasmic tail (8x/HXB-TM). The parental HXB and 8x Envs were CD4 dependent and independent, respectively, as expected, and HXB-fs, while exhibiting enhanced fusion, remained CD4 dependent (28, 29). However, restoring a full-length CT to 8x considerably reduced both the fusion efficiency and CD4 independence.


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DISCUSSION
 
A long CT of approximately 150 amino acids is a hallmark of most lentiviral envelope glycoproteins, including those of HIV-1, HIV-2, SIV, equine infectious anemia virus, and visna virus. Although the evolutionary importance of a long CT is uncertain, it is clear that the CT can modulate Env fusion levels, expression on the cell surface, and incorporation into viral particles (15, 33, 35, 55, 61, 80, 81, 88, 100). Although some of these effects may be cell type dependent (63), for HIV-1, HIV-2, and SIV, CT truncations can increase Env fusogenicity and surface expression (61, 90, 97, 100), while Env incorporation into virions can be increased or decreased (33, 35, 97). For the lab-derived CD4-independent isolate 8x, a CT truncation resulting from an fs mutation at the junction between the membrane-spanning domain and the CT was necessary for CD4-independent fusion on CXCR4, although additional mutations in gp120 that flanked the coreceptor binding site were also required (28, 29, 49). However, the fs mutation or a stop codon that generated a prematurely truncated tail of the same length was shown to produce striking changes in the Env ectodomain, increasing the fusogenicity and exposing gp120 epitopes that overlapped the conserved chemokine receptor binding domain on the gp120 core (28, 29). The mechanism by which this CT truncation produces these functional and structural changes has remained unclear, as have the determinants in the transmembrane domain (TM) that are involved.

In the present report, we show that for the HXBc2 Env, CTs truncated at or proximal to the start of the LLP-2 domain (amino acids 770 to 795), the most proximal of three alpha-helical domains in CT, increased Env fusion kinetics two- to fourfold. This increase was observed for Envs truncated at Ile-733, Leu-753, a palmitoylation site at Cys-764, and Leu-771 at the start of LLP-2, but not for an Env truncated at position 808, distal to LLP-2 and before the start of LLP-1 (amino acids 828 to 856) (Fig. 1). This effect could not be explained by differences in Env surface expression and was seen for X4- and R5-tropic Envs on CXCR4- and CCR5-expressing target cells. Although LLP-2 has been implicated in binding to several intracellular proteins, including calmodulin (6, 60, 82, 85), {alpha}-catenin (44), p115-RhoGEF (98), and the prenylated Rab acceptor protein (98), substantial structural and biochemical evidence indicates that this domain likely interacts with the inner surface of the plasma membrane (4, 18, 30, 37, 43, 45, 58, 83, 87, 94, 96). We also found that these mutations induced similar, although not identical, effects on the magnitude of cell fusion and the exposure of CD4-induced epitopes. Although stop codons at positions 733, 753, and 764 increased the magnitude of fusion, the exposure of CD4-induced epitopes, and the fusion kinetics, a stop codon at Leu-771 at the start of LLP-2 increased the fusion kinetics without exposing CD4-induced epitopes or increasing fusion levels. It is possible that fusion kinetics is a more sensitive indicator of qualitative changes in Env function than are assays for 17b and 48d epitope exposure and the absolute level of fusion activity. Nonetheless, collectively our findings are consistent with the view that the association of LLP-2 with the plasma membrane and/or interactions with one or more cellular proteins alter the conformation of the Env ectodomain to reduce Env fusogenicity and that this effect can be overcome by mutations that eliminate this domain.

The finding that a distal, membrane-interactive element in the HIV-1 CT can reduce Env fusogenicity is highly reminiscent of the case for other retrovirus Envs, including those of murine leukemia viruses, Mason-Pfizer monkey virus, and gibbon ape leukemia virus. For these Envs, cleavage of the CT C terminus by a viral protease is required for maximal fusion activity (12, 13, 20, 41, 70, 74). C-terminal cleavage of the Env CT has also been seen for equine infectious anemia virus Env (75), although not for those of primate lentiviruses (90). For Moloney murine leukemia virus, which has a CT of 32 amino acids, the cleaved C-terminal 16 amino acids, the R peptide, is predicted to form an amphipathic helix and to adopt a conformation that, similar to that of LLP-2, may facilitate a membrane interaction (84). This interaction may also be facilitated by an as yet unidentified palmitoylation signal (65). For Mason-Pfizer monkey virus, gibbon ape leukemia virus, and murine leukemia virus, cleavage of the CT C terminus occurs during virion maturation and is required to generate a fully infectious and fusogenic particle (13, 14, 20, 41, 93, 99). Indeed, the introduction of an R peptide onto the C terminus of a truncated CT of a simian immunodeficiency virus was shown to reduce Env fusogenicity on some, though not all, chemokine receptors (93, 95). Interestingly, although primate lentiviral CTs are not cleaved, Gag processing during virion maturation is closely coupled to Env function in that proteolytic cleavage of the p55 Gag precursor protein is required to generate maximal Env fusion activity (62, 90). Virions lacking Gag cleavage sites were reduced two- to threefold in fusion activity, and this effect was not seen when Envs with truncated CTs were used. Along with evidence that gp41 is more stably associated on virions before Gag is cleaved than afterwards (91) and the results of several reports implicating interactions between the gp41 CT and Gag matrix proteins (15, 32, 33, 54, 91), these findings strongly suggest that an association between the HIV CT and Gag p55 limits Env fusogenicity until Gag is fully processed. Our present findings add to this model by showing that even in the absence of Gag, Env fusion activity is restrained by its CT, and in particular, by a region that includes the LLP-2 domain. Thus, even prior to its association with Gag at the site of virion assembly, the CT limits Env fusion and ensures that full fusogenic activity is not generated until viral particles are formed and released. As previously suggested, for many enveloped viruses the Env CT may have evolved to protect infected cells from Env-induced cytopathic effects that could impede the production and/or assembly of progeny virions (62, 69, 74, 90).

Although our studies have implicated LLP-2 in restricting Env fusogenicity, the underlying mechanism for this "inside-out" signaling effect is unclear. Increased fusion efficiency, accelerated fusion kinetics, and the exposure of CD4-induced epitopes on Envs truncated prior to LLP-2 are all consistent with conformational changes in the Env ectodomain. This possibility was also suggested by studies with SIV showing biochemical differences between Envs with long versus prematurely truncated CTs (81). Similar findings have been observed for murine leukemia virus, where removal of the R peptide altered biochemical and immunological properties of the Env ectodomain (3). For both murine leukemia virus (3) and HIV-1 (Fig. 6) (28, 29, 49), these qualitative differences in Env could not be accounted for by differences in Env surface expression. The loss of a membrane anchor in the CT could affect the mobility and/or recruitment of Env trimers during formation and expansion of the fusion pore (56, 57, 90). In addition, the loss of this element could alter the distribution of Env on the cell surface in microdomains such as lipid rafts and/or or its association with other cell surface molecules (22). Alternatively, the loss of a CT membrane anchor could affect the stability of the gp120-gp41 association and its transition to a fusion-active conformation. Indeed, a recent report by Abrahamyan observed that the HIV-1 gp41 CT could slow the formation of the final stage of the gp41 six-helix bundle during fusion (1).

Whatever the mechanism, it is clear that for HIV, a loss of the distal CT can have profound effects on coreceptor utilization. Aside from changes in kinetics and fusion efficiency, the TM truncation was necessary, although not sufficient, to enable 8x to utilize CXCR4 in the absence of CD4 (49). Although not all CD4-independent viruses require a truncated CT (25, 26, 46), this change, possibly through its effects on kinetics and/or conformational changes in the ectodomain, can apparently complement mutations in gp120 to promote CD4 independence. Notably, although Envs with tails truncated at amino acids 733, 753, and 764 exposed CD4-induced epitopes on gp120, this effect was not sufficient for CD4 independence (Fig. 3 and 8) (28, 29). Similarly, the more rapid fusion kinetics observed for HXBc2 Envs with CTs truncated at or before position 771 could not be explained simply by the exposure of CD4-induced epitopes, since the 771* construct exhibited accelerated fusion without exposing these epitopes. Interestingly, when the effects of a CT truncation were evaluated with the dual-tropic 89.6 virus, fusion kinetics were increased to a greater extent on CCR5- than on CXCR4-expressing cells, suggesting that conformational changes and/or increased mobilities of Env proteins had a greater impact on CCR5 than on CXCR4 usage. Alternatively, these differences could have resulted at least in part from the higher affinity of Envs for CCR5 than for CXCR4 binding, since coreceptor affinity has been shown to contribute substantially to fusion kinetics (71, 73).

CT truncations increase the sensitivity of HIV-1 strains to neutralization by sera from infected individuals, an effect likely due at least in part to the increased exposure of CD4-induced epitopes on gp120 and/or possibly gp41 (29). Interestingly, this effect occurred in spite of the more rapid fusion kinetics of these Envs, which might have been expected to render them more neutralization resistant (73). However, 8x is more resistant to the entry inhibitor ENF (36), a peptide based on the gp41 HR2 sequence that blocks the association of HR2 with HR1 during formation of the six-helix bundle (17, 34, 57). ENF resistance was attributed to the more rapid fusion kinetics of 8x (36), although in this study the possibility was not ruled out that mutations in the 8x ectodomain, including those in HR1, could have been involved (49). In the present study, we show for X4-tropic HXBc2 and R5-tropic JRFL that a CT truncation alone is sufficient to render these Envs more resistant to ENF (Fig. 7). Similar findings were recently reported, although cytoplasmic domains involved in mediating this effect were not identified (2). Collectively, these findings are consistent with the view that more rapid fusion kinetics decreases the time of exposure of the fusion intermediate conformation on gp41 (36, 71, 73), thereby reducing the sensitivity to ENF inhibition.

In summary, we showed that the CT of HIV-1 gp41, in particular a region that includes the LLP-2 domain, reduces Env fusogenicity through a process that involves conformational changes in the Env ectodomain. Along with evidence that the association of the CT with the Gag precursor protein also limits Env fusogenicity on virions until Gag proteolytic processing is completed (62), these findings support a model in which the gp41 CT tightly regulates Env function during its transport to sites of virion assembly, both before associating with Gag and afterwards during virion formation. Thus, the Env fusion function is not fully enabled until mature viral particles are formed and released from cells. This property is shared by a number of enveloped viruses and may serve as a general mechanism to reduce Env-induced cytopathic effects on virus-producing cells, thereby facilitating virus production. How interactions of the HIV CT with membranes and/or cellular molecules alter the conformation of the Env ectodomain, how these interactions directly affect the formation of the fusion pore, and how the CT continues to exert this effect during its transfer from a cellular environment to a virion will be important areas for further investigation.


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ACKNOWLEDGMENTS
 
This work was supported by NIH grant RO1 AI45378 to J.A.H. and by grants from the Swiss National Science Foundation to S.W. and F.B. (grants 823A-064728 and 823A-61172, respectively). This work was made possible with the support of the Viral Cell Molecular Core of the Penn Center for AIDS Research (NIH P30 AI45008). This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

We gratefully thank James Robinson (Tulane University) for providing monoclonal antibodies to HIV-1 gp120 and Trimeris Inc. (Durham, NC) for providing enfuvirtide. We also thank Robert Doms and George Lin for helpful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of Pennsylvania, Rm. 356, Biomedical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-0261. Fax: (215) 573-7356. E-mail: hoxie{at}mail.med.upenn.edu. Back


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REFERENCES
 
    1
  1. Abrahamyan, L. G., R. M. Markosyan, J. P. Moore, F. S. Cohen, and G. B. Melikyan. 2003. Human immunodeficiency virus type 1 Env with an intersubunit disulfide bond engages coreceptors but requires bond reduction after engagement to induce fusion. J. Virol. 77:5829-5836.[Abstract/Free Full Text]
    2. 2
  2. Abrahamyan, L. G., S. R. Mkrtchyan, J. Binley, M. Lu, G. B. Melikyan, and F. S. Cohen. 2005. The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle. J. Virol. 79:106-115.[Abstract/Free Full Text]
    3. 3
  3. Aguilar, H. C., W. F. Anderson, and P. M. Cannon. 2003. Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the R peptide. J. Virol. 77:1281-1291.
    4. 4
  4. Andreassen, H., H. Bohr, J. Bohr, S. Brunak, T. Bugge, R. M. Cotterill, C. Jacobsen, P. Kusk, B. Lautrup, S. B. Petersen, et al. 1990. Analysis of the secondary structure of the human immunodeficiency virus (HIV) proteins p17, gp120, and gp41 by computer modeling based on neural network methods. J. Acquir. Immune. Defic. Syndr. 3:615-622.
    5. 5
  5. Babcock, G. J., M. Farzan, and J. Sodroski. 2003. Ligand-independent dimerization of CXCR4, a principal HIV-1 coreceptor. J. Biol. Chem. 278:3378-3385.[Abstract/Free Full Text]
    6. 6
  6. Beary, T. P., S. B. Tencza, T. A. Mietzner, and R. C. Montelaro. 1998. Interruption of T-cell signal transduction by lentivirus lytic peptides from HIV-1 transmembrane protein. J. Pept. Res. 51:75-79.[Medline]
    7. 7
  7. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657-700.[CrossRef][Medline]
    8. 8
  8. Berlioz-Torrent, C., B. L. Shacklett, L. Erdtmann, L. Delamarre, I. Bouchaert, P. Sonigo, M. C. Dokhelar, and R. Benarous. 1999. Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins. J. Virol. 73:1350-1361.[Abstract/Free Full Text]
    9. 9
  9. Bhattacharya, J., P. J. Peters, and P. R. Clapham. 2004. Human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles. J. Virol. 78:5500-5506.[Abstract/Free Full Text]
    10. 10
  10. Boge, M., S. Wyss, J. S. Bonifacino, and M. Thali. 1998. A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adaptor. J. Biol. Chem. 273:15773-15778.[Abstract/Free Full Text]
    11. 11
  11. Bowers, K., A. Pelchen-Matthews, S. Höning, P. J. Vance, L. Creary, B. S. Haggarty, J. Romano, W. Ballensiefen, J. A. Hoxie, and M. Marsh. 2000. The simian immunodeficiency virus envelope glycoprotein contains multiple signals that regulate its cell surface expression and endocytosis. Traffic 1:661-674.[CrossRef][Medline]
    12. 12
  12. Brody, B. A., and E. Hunter. 1992. Mutations within the env gene of Mason-Pfizer monkey virus: effects on protein transport and SU-TM association. J. Virol. 66:3466.[Abstract/Free Full Text]
    13. 13
  13. Brody, B. A., S. S. Rhee, and E. Hunter. 1994. Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. J. Virol. 68:4620-4627.[Abstract/Free Full Text]
    14. 14
  14. Brody, B. A., S. S. Rhee, M. A. Sommerfelt, and E. Hunter. 1992. A viral protease-mediated cleavage of the transmembrane glycoprotein of Mason-Pfizer monkey virus can be suppressed by mutations within the matrix protein. Proc. Natl. Acad. Sci. USA 89:3443-3447.[Abstract/Free Full Text]
    15. 15
  15. Celma, C. C., J. M. Manrique, J. L. Affranchino, E. Hunter, and S. A. Gonzalez. 2001. Domains in the simian immunodeficiency virus gp41 cytoplasmic tail required for envelope incorporation into particles. Virology 283:253-261.[CrossRef][Medline]
    16. 16
  16. Chakrabarti, S., J. R. Sisler, and B. Moss. 1997. Compact, synthetic, vaccinia virus early/late promoter for protein expression. BioTechniques 23:1094-1097.[Medline]
    17. 17
  17. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273.[CrossRef][Medline]
    18. 18
  18. Chen, S. S., S. F. Lee, and C. T. Wang. 2001. Cellular membrane-binding ability of the C-terminal cytoplasmic domain of human immunodeficiency virus type 1 envelope transmembrane protein gp41. J. Virol. 75:9925-9938.[Abstract/Free Full Text]
    19. 19
  19. Chernomordik, L., A. N. Chanturiya, E. Suss-Toby, E. Nora, and J. Zimmerberg. 1994. An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes. J. Virol. 68:7115-7123.[Abstract/Free Full Text]
    20. 20
  20. Christodoulopoulos, I., and P. M. Cannon. 2001. Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors. J. Virol. 75:4129-4138.[Abstract/Free Full Text]
    21. 21
  21. Comardelle, A. M., C. H. Norris, D. R. Plymale, P. J. Gatti, B. Choi, C. D. Fermin, A. M. Haislip, S. B. Tencza, T. A. Mietzner, R. C. Montelaro, and R. F. Garry. 1997. A synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of Xenopus laevis oocytes. AIDS Res. Hum. Retrovir. 13:1525-1532.[Medline]
    22. 22
  22. Deschambeault, J., J. P. Lalonde, G. Cervantes-Acosta, R. Lodge, E. A. Cohen, and G. LeMay. 1999. Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission. J. Virol. 73:5010-5017.[Abstract/Free Full Text]
    23. 23
  23. Dimitrov, A. S., S. S. Rawat, S. Jiang, and R. Blumenthal. 2003. Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion. Biochemistry 42:14150-14158.[CrossRef][Medline]
    24. 24
  24. Doms, R. W. 2001. Chemokine receptors and HIV entry. AIDS 15(Suppl. 1):S34-S35.
    25. 25
  25. Dumonceaux, J., C. Goujon, V. Joliot, P. Briand, and U. Hazan. 2001. Determination of essential amino acids involved in the CD4-independent tropism of the X4 human immunodeficiency virus type 1 m7NDK isolate: role of potential N glycosylations in the C2 and V3 regions of gp120. J. Virol. 75:5425-5428.[Abstract/Free Full Text]
    26. 26
  26. Dumonceaux, J., S. Nisole, C. Chanel, L. Quivet, A. Amara, F. Baleux, P. Briand, and U. Hazan. 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]
    27. 27
  27. Edinger, A. L., J. L. Mankowski, B. J. Doranz, B. J. Margulies, B. Lee, J. Rucker, M. Sharron, T. L. Hoffman, J. F. Berson, M. C. Zink, V. M. Hirsch, J. E. Clements, and R. W. Doms. 1997. CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain. Proc. Natl. Acad. Sci. USA 94:14742-14747.[Abstract/Free Full Text]
    28. 28
  28. Edwards, T. G., T. L. Hoffman, F. Baribaud, S. Wyss, C. C. LaBranche, J. Romano, J. Adkinson, M. Sharron, J. A. Hoxie, and R. W. Doms. 2001. Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein. J. Virol. 75:5230-5239.[Abstract/Free Full Text]
    29. 29
  29. Edwards, T. G., S. Wyss, J. D. Reeves, S. Zolla-Pazner, J. A. Hoxie, R. W. Doms, and F. Baribaud. 2002. Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein. J. Virol. 76:2683-2691.[Abstract/Free Full Text]
    30. 30
  30. Eisenberg, D., and M. Wesson. 1990. The most highly amphiphilic alpha-helices include two amino acid segments in human immunodeficiency virus glycoprotein 41. Biopolymers 29:171-177.[CrossRef][Medline]
    31. 31
  31. Endres, M. J., P. R. Clapham, M. Marsh, M. Ahuja, J. D. Turner, A. McKnight, J. F. Thomas, B. Stoebenau-Haggarty, S. Choe, P. J. Vance, T. N. Wells, C. A. Power, S. S. Sutterwala, R. W. Doms, N. R. Landau, and J. A. Hoxie. 1996. CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87:745-756.[CrossRef][Medline]
    32. 32
  32. Freed, E. O., and M. A. Martin. 1996. Domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions. J. Virol. 70:341-351.[Abstract]
    33. 33
  33. Freed, E. O., and M. A. Martin. 1995. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J. Virol. 69:1984-1989.[Abstract]
    34. 34
  34. Furuta, R. A., C. T. Wild, Y. Weng, and C. D. Weiss. 1998. Capture of an early fusion-active conformation of HIV-1 gp41. Nat. Struct. Biol. 5:276-279. (Erratum, 5:612.)
    35. 35
  35. Gabuzda, D. H., A. Lever, E. Terwilliger, and J. Sodroski. 1992. Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 66:3306-3315.[Abstract/Free Full Text]
    36. 36
  36. Gallo, S. A., A. Puri, and R. Blumenthal. 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]
    37. 37
  37. Gawrisch, K., K. H. Han, J. S. Yang, L. D. Bergelson, and J. A. Ferretti. 1993. Interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type I with lipid bilayers. Biochemistry 32:3112-3118.[CrossRef][Medline]
    38. 38
  38. Hoffman, T. L., C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms. 1999. Stable exposure of the coreceptor binding site in a CD4-independent HIV-1 envelope protein. Proc. Natl. Acad. Sci. USA 96:6359-6364.[Abstract/Free Full Text]
    39. 39
  39. Hug, P., H. M. Lin, T. Korte, X. Xiao, D. S. Dimitrov, J. M. Wang, A. Puri, and R. Blumenthal. 2000. Glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing CD4, CXCR4, and/or CCR5. J. Virol. 74:6377-6385.[Abstract/Free Full Text]
    40. 40
  40. Ishikawa, H., M. Sasaki, S. Noda, and Y. Koga. 1998. Apoptosis induction by the binding of the carboxyl terminus of human immunodeficiency virus type 1 gp160 to calmodulin. J. Virol. 72:6574-6580.[Abstract/Free Full Text]
    41. 41
  41. Januszeski, M. M., P. M. Cannon, D. Chen, Y. Rozenberg, and W. F. Anderson. 1997. Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein. J. Virol. 71:3613-3619.[Abstract]
    42. 42
  42. Jones, P. L., T. Korte, and R. Blumenthal. 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]
    43. 43
  43. Kalia, V., S. Sarkar, P. Gupta, and R. C. Montelaro. 2003. Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation. J. Virol. 77:3634-3646.[Abstract/Free Full Text]
    44. 44
  44. Kim, E. M., K. H. Lee, and J. W. Kim. 1999. The cytoplasmic domain of HIV-1 gp41 interacts with the carboxyl-terminal region of alpha-catenin. Mol. Cell 9:281-285.
    45. 45
  45. Kliger, Y., and Y. Shai. 1997. A leucine zipper-like sequence from the cytoplasmic tail of the HIV-1 envelope glycoprotein binds and perturbs lipid bilayers. Biochemistry 36:5157-5169.[CrossRef][Medline]
    46. 46
  46. Kolchinsky, P., E. Kiprilov, P. Bartley, R. Rubinstein, and J. Sodroski. 2001. Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops. J. Virol. 75:3435-3443.[Abstract/Free Full Text]
    47. 47
  47. Kolchinsky, P., E. Kiprilov, and J. Sodroski. 2001. Increased neutralization sensitivity of CD4-independent human immunodeficiency virus variants. J. Virol. 75:2041-2050.[Abstract/Free Full Text]
    48. 48
  48. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature (London) 393:648-659.[CrossRef][Medline]
    49. 49
  49. LaBranche, C. C., T. L. Hoffman, J. Romano, B. S. Haggarty, T. G. Edwards, T. J. Matthews, R. W. Doms, and J. A. Hoxie. 1999. Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity. J. Virol. 73:10310-10319.[Abstract/Free Full Text]
    50. 50
  50. LaBranche, C. C., M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, M. Marsh, and J. A. Hoxie. 1995. A single amino acid change in the cytoplasmic domain of SIVmac transmembrane molecule increases envelope glycoprotein expression on cells and virions. J. Virol. 69:5217-5227.[Abstract]
    51. 51
  51. Lin, G., and J. A. Hoxie. 2003. CCR5 mimicry by sulfated human anti-HIV-1 antibodies. Cell 114:147-148.[CrossRef][Medline]
    52. 52
  52. Lin, G., B. Lee, B. S. Haggarty, R. W. Doms, and J. A. Hoxie. 2001. CD4-independent use of rhesus CCR5 by human immunodeficiency virus type 2 implicates an electrostatic interaction between the CCR5 N terminus and the gp120 C4 domain. J. Virol. 75:10766-10778.[Abstract/Free Full Text]
    53. 53
  53. Lodge, R., J. P. Lalonde, G. LeMay, and E. A. Cohen. 1997. The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J. 16:695-705.[CrossRef][Medline]
    54. 54
  54. Mammano, F., E. Kondo, J. Sodroski, A. Bukovsky, and H. G. Göttlinger. 1995. Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains. J. Virol. 69:3824-3830.[Abstract]
    55. 55
  55. Manrique, J. M., C. C. Celma, J. L. Affranchino, E. Hunter, and S. A. Gonzalez. 2001. Small variations in the length of the cytoplasmic domain of the simian immunodeficiency virus transmembrane protein drastically affect envelope incorporation and virus entry. AIDS Res. Hum. Retrovir. 17:1615-1624.[CrossRef][Medline]
    56. 56
  56. Melikyan, G. B., R. M. Markosyan, S. A. Brener, Y. Rozenberg, and F. S. Cohen. 2000. Role of the cytoplasmic tail of ecotropic Moloney murine leukemia virus Env protein in fusion pore formation. J. Virol. 74:447-455.[Abstract/Free Full Text]
    57. 57
  57. Melikyan, G. B., R. M. Markosyan, H. Hemmati, M. K. Delmedico, D. M. Lambert, and F. S. Cohen. 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]
    58. 58
  58. Miller, M. A., M. W. Cloyd, J. Liebmann, C. R. Rinaldo, Jr., K. R. Islam, S. Z. Wang, T. A. Mietzner, and R. C. Montelaro. 1993. Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein. Virology 196:89-100.[CrossRef][Medline]
    59. 59
  59. Miller, M. A., R. F. Garry, J. M. Jaynes, and R. C. Montelaro. 1991. A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides. AIDS Res. Hum. Retrovir. 7:511-519.[Medline]
    60. 60
  60. Miller, M. A., T. A. Mietzner, M. W. Cloyd, W. G. Robey, and R. C. Montelaro. 1993. Identification of a calmodulin-binding and inhibitory peptide domain in the HIV-1 transmembrane glycoprotein. AIDS Res. Hum. Retrovir. 9:1057-1066.[Medline]
    61. 61
  61. Mulligan, M. J., G. V. Yamshchikov, G. D. Ritter, Jr., F. Gao, M. J. Jin, C. D. Nail, C. P. Spies, B. H. Hahn, and R. W. Compans. 1992. Cytoplasmic domain truncation enhances fusion activity by the exterior glycoprotein complex of human immunodeficiency virus type 2 in selected cell types. J. Virol. 66:3971-3975.[Abstract/Free Full Text]
    62. 62
  62. Murakami, T., S. Ablan, E. O. Freed, and Y. Tanaka. 2004. Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity. J. Virol. 78:1026-1031.[Abstract/Free Full Text]
    63. 63
  63. Murakami, T., and E. O. Freed. 2000. The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions. Proc. Natl. Acad. Sci. USA 97:343-348.[Abstract/Free Full Text]
    64. 64
  64. Ohno, H., R. C. Aguilar, M. C. Fournier, S. Hennecke, P. Cosson, and J. S. Bonifacino. 1997. Interaction of endocytic signals from the HIV-1 envelope glycoprotein complex with members of the adaptor medium chain family. Virology 238:305-315.[CrossRef][Medline]
    65. 65
  65. Olsen, K. E., and K. B. Andersen. 1999. Palmitoylation of the intracytoplasmic R peptide of the transmembrane envelope protein in Moloney murine leukemia virus. J. Virol. 73:8975-8981.[Abstract/Free Full Text]
    66. 66
  66. Owens, R. J., J. W. Dubay, E. Hunter, and R. W. Compans. 1991. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88:3987-3991.[Abstract/Free Full Text]
    67. 67
  67. Pierson, T. C., and R. W. Doms. 2003. HIV-1 entry and its inhibition. Curr. Top. Microbiol. Immunol. 281:1-27.[Medline]
    68. 68
  68. Puffer, B. A., S. Pohlmann, A. L. Edinger, D. Carlin, M. D. Sanchez, J. Reitter, D. D. Watry, H. S. Fox, R. C. Desrosiers, and R. W. Doms. 2002. CD4 independence of simian immunodeficiency virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity. J. Virol. 76:2595-2605.[Abstract/Free Full Text]
    69. 69
  69. Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220-3231.[Abstract/Free Full Text]
    70. 70
  70. Ragheb, J. A., and W. F. Anderson. 1994. Uncoupled expression of Moloney murine leukemia virus envelop polypeptides SU and TM: a functional analysis of the role of TM domains in viral entry. J. Virol. 268:3207-3219.
    71. 71
  71. Reeves, J. D., S. A. Gallo, N. Ahmad, J. L. Miamidian, P. E. Harvey, M. Sharron, S. Pohlmann, J. N. Sfakianos, C. A. Derdeyn, R. Blumenthal, E. Hunter, and R. W. Doms. 2002. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc. Natl. Acad. Sci. USA 99:16249-16254.[Abstract/Free Full Text]
    72. 72
  72. Reeves, J. D., S. Hibbitts, G. Simmons, A. McKnight, J. M. Azevedo-Pereira, J. Moniz-Pereira, and P. R. Clapham. 1999. Primary human immunodeficiency virus type 2 (HIV-2) isolates infect CD4-negative cells via CCR5 and CXCR4: comparison with HIV-1 and simian immunodeficiency virus and relevance to cell tropism in vivo. J. Virol. 73:7795-7804.[Abstract/Free Full Text]
    73. 73
  73. Reeves, J. D., F.-H. Lee, J. L. Miamidian, C. B. Jabara, M. Juntilla, and R. W. Doms. 2005. Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity and virus neutralization. J. Virol. 79:4991-4999.[Abstract/Free Full Text]
    74. 74
  74. Rein, A., J. Mirro, J. G. Haynes, S. M. Ernst, and K. Nagashima. 1994. Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68:1773-1781.[Abstract/Free Full Text]
    75. 75
  75. Rice, N. R., L. E. Henderson, R. C. Sowder, T. D. Copeland, S. Oroszlan, and J. F. Edwards. 1990. Synthesis and processing of the transmembrane envelope protein of equine infectious anemia virus. J. Virol. 64:3770-3778.[Abstract/Free Full Text]
    76. 76
  76. Rousso, I., M. B. Mixon, B. K. Chen, and P. S. Kim. 2000. Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. Proc. Natl. Acad. Sci. USA 97:13523-13525.[Abstract/Free Full Text]
    77. 77
  77. Rowell, J. F., P. E. Stanhope, and R. F. Siliciano. 1995. Endocytosis of the HIV-1 envelope protein: mechanism and role in processing for association with class II MHC. J. Immunol. 155:473-488.[Abstract]
    78. 78
  78. Rucker, J., B. J. Doranz, A. L. Edinger, D. Long, J. F. Berson, and R. W. Doms. 1997. Cell-cell fusion assay to study role of chemokine receptors in human immunodeficiency virus type 1 entry. Methods Enzymol. 288:118-133.[Medline]
    79. 79
  79. Sauter, M. M., A. Pelchen-Matthews, R. Bron, M. Marsh, C. C. LaBranche, P. J. Vance, J. Romano, B. S. Haggarty, T. K. Hart, W. M. F. Lee, and J. A. Hoxie. 1996. An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface. J. Cell Biol. 132:795-811.[Abstract/Free Full Text]
    80. 80
  80. Spies, C. P., and R. W. Compans. 1994. Effects of cytoplasmic domain length on cell surface expression and syncytium-forming capacity of the simian immunodeficiency virus envelope glycoprotein. Virology 203:8-19.[CrossRef][Medline]
    81. 81
  81. Spies, C. P., G. D. Ritter, Jr., M. J. Mulligan, and R. W. Compans. 1994. Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein alters the conformation of the external domain. J. Virol. 68:585-591.[Abstract/Free Full Text]
    82. 82
  82. Srinivas, S. K., R. V. Srinivas, G. M. Anantharamaiah, R. W. Compans, and J. P. Segrest. 1993. Cytosolic domain of the human immunodeficiency virus envelope glycoproteins binds to calmodulin and inhibits calmodulin-regulated proteins. J. Biol. Chem. 268:22895-22899.[Abstract/Free Full Text]
    83. 83
  83. Srinivas, S. K., R. V. Srinivas, G. M. Anantharamaiah, J. P. Segrest, and R. W. Compans. 1992. Membrane interactions of synthetic peptides corresponding to amphipathic helical segments of the human immunodeficiency virus type-1 envelope glycoprotein. J. Biol. Chem. 267:7121-7127.[Abstract/Free Full Text]
    84. 84
  84. Taylor, G. M., and D. A. Sanders. 2003. Structural criteria for regulation of membrane fusion and virion incorporation by the murine leukemia virus TM cytoplasmic domain. Virology 312:295-305.[CrossRef][Medline]
    85. 85
  85. Tencza, S. B., T. A. Mietzner, and R. C. Montelaro. 1997. Calmodulin-binding function of LLP segments from the HIV type 1 transmembrane protein is conserved among natural sequence variants. AIDS Res. Hum. Retrovir. 13:263-269.[Medline]
    86. 86
  86. Thali, M., J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski. 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J. Virol. 67:3978-3988.[Abstract/Free Full Text]
    87. 87
  87. Venable, R. M., R. W. Pastor, B. R. Brooks, and F. W. Carson. 1989. Theoretically determined three-dimensional structures for amphipathic segments of the HIV-1 gp41 envelope protein. AIDS Res. Hum. Retrovir. 5:7-22.[Medline]
    88. 88
  88. Wilk, T., T. Pfeiffer, and V. Bosch. 1992. Retained in vitro infectivity and cytopathogenicity of HIV-1 despite truncation of the C-terminal tail of the env gene product. Virology 189:167-177.[CrossRef][Medline]
    89. 89
  89. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature (London) 393:705-711.[CrossRef][Medline]
    90. 90
  90. Wyma, D. J., J. Jiang, J. Shi, J. Zhou, J. E. Lineberger, M. D. Miller, and C. Aiken. 2004. Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail. J. Virol. 78:3429-3435.[Abstract/Free Full Text]
    91. 91
  91. Wyma, D. J., A. Kotov, and C. Aiken. 2000. Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles. J. Virol. 74:9381-9387.[Abstract/Free Full Text]
    92. 92
  92. Wyss, S., C. Berlioz-Torrent, M. Boge, G. Blot, S. Honing, R. Benarous, and M. Thali. 2001. The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin J. Virol. 75:2982-2992. (Erratum, 75:4473.)[Free Full Text]
    93. 93
  93. Yang, C., and R. W. Compans. 1997. Analysis of the murine leukemia virus R peptide: delineation of the molecular determinants which are important for its fusion inhibition activity. J. Virol. 71:8490-8496.[Abstract]
    94. 94
  94. Yang, C., C. P. Spies, and R. W. Compans. 1995. The human and simian immunodeficiency virus envelope glycoprotein transmembrane subunits are palmitoylated. Proc. Natl. Acad. Sci. USA 92:9871-9875.[Abstract/Free Full Text]
    95. 95
  95. Yang, C., Q. Yang, and R. W. Compans. 2000. Coreceptor-dependent inhibition of the cell fusion activity of simian immunodeficiency virus Env proteins. J. Virol. 74:6217-6222.[Abstract/Free Full Text]
    96. 96
  96. Yuan, T., S. Tencza, T. A. Mietzner, R. C. Montelaro, and H. J. Vogel. 2001. Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41. Biopolymers 58:50-62.[CrossRef][Medline]
    97. 97
  97. Yuste, E., J. D. Reeves, R. W. Doms, and R. C. Desrosiers. 2004. Modulation of Env content in virions of simian immunodeficiency virus: correlation with cell surface expression and virion infectivity. J. Virol. 78:6775-6785.[Abstract/Free Full Text]
    98. 98
  98. Zhang, H., L. Wang, S. Kao, I. P. Whitehead, M. J. Hart, B. Liu, K. Duus, K. Burridge, C. J. Der, and L. Su. 1999. Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF. Curr. Biol. 9:1271-1274.[CrossRef][Medline]
    99. 99
  99. Zhao, Y., L. Zhu, C. A. Benedict, D. Chen, W. F. Anderson, and P. M. Cannon. 1998. Functional domains in the retroviral transmembrane protein. J. Virol. 72:5392-5398.[Abstract/Free Full Text]
    100. 100
  100. Zingler, K., and D. R. Littman. 1993. Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases Env incorporation into particles and fusogenicity and infectivity. J. Virol. 67:2824-2831.[Abstract/Free Full Text]

Journal of Virology, October 2005, p. 12231-12241, Vol. 79, No. 19
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.19.12231-12241.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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