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

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.     

Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail

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.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

Effect of extension of the cytoplasmic domain of human immunodeficiency type 1 virus transmembrane protein gp41 on virus replication

The biological significance of the presence of a long cytoplasmic domain in the envelope (Env) transmembrane protein gp41 of human immunodeficiency virus type 1 (HIV-1) is still not fully understood. Here we examined the effects of cytoplasmic tail elongation on virus replication and characterized the role of the C-terminal cytoplasmic tail in interactions with the Gag protein. Extensions with six and nine His residues but not with fewer than six His residues were found to severely inhibit virus replication through decreased Env electrophoretic mobility and reduced Env incorporation compared to the wild-type virus. These two mutants also exhibited distinct N glycosylation and reduced cell surface expression. An extension of six other residues had no deleterious effect on infectivity, even though some mutants showed reduced Env incorporation into the virus and/or decreased cell surface expression. We further show that these elongated cytoplasmic tails in a format of the glutathione S-transferase fusion protein still interacted effectively with the Gag protein. In addition, the immediate C terminus of the cytoplasmic tail was not directly involved in interactions with Gag, but the region containing the last 13 to 43 residues from the C terminus was critical for Env-Gag interactions. Taken together, our results demonstrate that HIV-1 Env can tolerate extension at its C terminus to a certain degree without loss of virus infectivity and Env-Gag interactions. However, extended elongation in the cytoplasmic tail may impair virus infectivity, Env cell surface expression, and Env incorporation into the virus.     

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.     

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.     

HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor (gp160) that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly, the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and coreceptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation and the role of specific membrane microdomains in this process. Here, we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.     

Photoinduced reactivity of the HIV-1 envelope glycoprotein with a membrane-embedded probe reveals insertion of portions of the HIV-1 Gp41 cytoplasmic tail into the viral membrane

The interactions of HIV-1 Env (gp120-gp41) with CD4 and coreceptors trigger a barrage of conformational changes in Env that drive the membrane fusion process. Various regions of gp41 have profound effects on HIV entry and budding. However, the precise interactions between gp41 and the membrane have not been elucidated. To examine portions of membrane proteins that are embedded in membrane lipids, we have studied photoinduced chemical reactions in membranes using the lipid bilayer specific probe iodonaphthyl azide (INA). Here we show that in addition to the transmembrane anchor, amphipatic sequences in the cytoplasmic tail (CT) of HIV-1 gp41 are labeled by INA. INA labeling of the HIV-1 gp41 CT was similar whether wild-type or a mutant HIV-1 was used with uncleaved p55 Gag, which does not allow entry. These results shed light on the disposition of the HIV-1 gp41 CT with respect to the membrane. Moreover, our data have general implications for topology of membrane proteins and their in situ interactions with the lipid bilayer.     

Surface exposure of the HIV-1 env cytoplasmic tail LLP2 domain during the membrane fusion process: interaction with gp41 fusion core

HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1-2), which includes two alpha-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1-2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 degrees C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1-2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion.     

Membrane Structure Correlates to Function of LLP2 on the Cytoplasmic Tail of HIV-1 gp41 Protein

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (−1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.     

Membrane Structure Correlates to Function of LLP2 on the Cytoplasmic Tail of HIV-1 gp41 Protein

Mutation studies previously showed that the lentivirus lytic peptide (LLP2) sequence of the cytoplasmic C-terminal tail of the HIV-1 gp41 envelope protein inhibited viral-initiated T-cell death and T-cell syncytium formation, at which time in the HIV life cycle the gp41 protein is embedded in the T-cell membrane. In striking contrast, the mutants did not affect virion infectivity, during which time the gp41 protein is embedded in the HIV envelope membrane. To examine the role of LLP2/membrane interactions, we applied synchrotron x-radiation to determine structure of hydrated membranes. We focused on WT LLP2 peptide (+3 charge) and MX2 mutant (−1 charge) with membrane mimics for the T-cell and the HIV-1 membranes. To investigate the influence of electrostatics, cholesterol content, and peptide palmitoylation, we also studied three other LLP2 variants and HIV-1 mimics without negatively charged lipids or cholesterol as well as extracted HIV-1 lipids. All LLP2 peptides bound strongly to T-cell membrane mimics, as indicated by changes in membrane structure and bending. In contrast, none of the weakly bound LLP2 variants changed the HIV-1 membrane mimic structure or properties. This correlates well with, and provides a biophysical basis for, previously published results that reported lack of a mutant effect in HIV virion infectivity in contrast to an inhibitory effect in T-cell syncytium formation. It shows that interaction of LLP2 with the T-cell membrane modulates biological function.     

Mutations in the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein impair the incorporation of Env proteins into mature virions.

In-frame stop codons were introduced into the coding region of human immunodeficiency virus type 1 (HIV-1) transmembrane protein (gp41). Truncation of 147 amino acids from the carboxyl terminus of gp41 (TM709) significantly decreased the stability and cell surface expression of the viral Env proteins, while truncation of 104 amino acids (TM752) did not. Truncation of 43 or more amino acids from the carboxyl terminus of gp41 generated mutant viruses which were noninfectious in several human CD4+ T lymphoid cell lines and fresh peripheral blood mononuclear cells. Analysis of the noninfectious mutant virions revealed significantly reduced incorporation of the Env proteins compared with the wild-type virions. Comparable amounts of Env proteins were detected on the surfaces of wild-type- and TM752-transfected cells, suggesting that the structures of gp41 required for efficient incorporation of Env proteins were disrupted in mutant TM752. Truncation of the last 12 amino acids (TM844) from the carboxyl terminus of gp41 did not significantly affect the assembly and release of virions or the incorporation of Env proteins into mature virions. However, the TM844 virus had dramatically decreased infectivity compared with the wild-type virus. This suggests that the cytoplasmic domain of gp41 also plays a role in other steps of virus replication.     

Cell-dependent requirement of human immunodeficiency virus type 1 gp41 cytoplasmic tail for Env incorporation into virions

Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.     

Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.     

The dominant-negative action of a fusion protein containing the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 in virus replication

We previously described a novel mode of downregulation of human immunodeficiency virus type 1 (HIV-1) Gag expression by a cytoplasmic domain fusion protein of the envelope (Env) transmembrane protein, β-galactosidase (β-gal)/706–856, which contains the cytoplasmic tail of gp41 fused at the C terminus of Escherichia coli β-gal. In the present study, we showed that this mediator conferred a dose-dependent dominant interference with virus infectivity. In the context of an HIV-1 provirus, this inhibitor downregulated steady-state Env expression. Paradoxically, Env overexpression suppressed β-gal/706–856-mediatd Gag downregulation. Sucrose gradient ultracentrifugation and confocal microscopy revealed that Gag, Env, and β-gal/706–856 had stable interactions and formed aggregated complexes in perinuclear regions. Moreover, Env overexpression hindered colocalization of Gag with β-gal/706–856 in the perinuclear region. Further cytoplasmic domain mapping analyses showed a correlation between the ability of cytoplasmic subdomains to downregulate Gag expression and the ability of these subdomains to stably interact with Gag. These studies show that redirection of Gag from its cytoplasmic synthesis site to a perinuclear compartment is a prerequisite for β-gal/706–856-mediated Gag downregulation. The results also illustrate that the dynamic interplay among Gag, Env, and β-gal/706–856 can modulate Gag and Env expression, thus controlling HIV-1 infection.     

Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion

The N-terminal fusion peptide and the interfacial sequence preceding the transmembrane anchor of HIV-1 gp41 are required for viral fusion. Studies with synthetic peptides indicated that these regions function by destabilizing membranes, which is regarded as a crucial step in the membrane fusion reaction. However, it is not clear whether membrane destabilization is induced by these sequences in the intact gp41. We address this question by examining fusion and destabilization of membranes expressing HIV-1(IIIB) wild-type Env and two mutant Envs. (1) A Glu residue at position 2 of the gp41 fusion peptide is substituted for Val (V2E) to produce one mutant. (2) Residues 665-682 in the membrane-proximal domain are deleted to form the other. The process of membrane destabilization was monitored by the influx of Sytox, an impermeant fluorescent dye, into the Env-expressing cells following the interaction with CD4-CXCR4 complexes, and fusion was monitored by observing dye transfer between Env-expressing cells and appropriate target cells. We also monitored the conformational changes in the Envs following their interactions with CD4 and CXCR4 by immunofluorescence using an anti-gp41 mAb that reacts with the six-helix bundle. In contrast to the wild type, both Env mutants did not mediate cell fusion. The V2E Env did not mediate membrane destabilization. However, the Env with an unmodified fusion peptide but with a deletion of residues 665-682 in the membrane-proximal domain did mediate membrane destabilization. The wild type and both mutant Envs undergo conformational changes detected by the anti-gp41 six-helix bundle mAbs. Our results suggest that in intact HIV-1 Env the membrane-proximal domain is not required for membrane perturbations, but rather enables the bending of gp41 that is required for viral and target membranes to come together. Moreover, the observation that the Delta665-683 Env self-inserts its fusion peptide but does not cause fusion suggests that self-insertion of the fusion peptide is not sufficient for HIV-1 Env-mediated fusion.     

The Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41 Harbors Lipid Raft Association Determinants

The molecular basis for localization of the human immunodeficiency virus type 1 envelope glycoprotein (Env) in detergent-resistant membranes (DRMs), also called lipid rafts, still remains unclear. The C-terminal cytoplasmic tail of gp41 contains three membrane-interacting, amphipathic α-helical sequences, termed lentivirus lytic peptide 2 (LLP-2), LLP-3, and LLP-1, in that order. Here we identify determinants in the cytoplasmic tail which are crucial for Env''s association with Triton X-100-resistant rafts. Truncations of LLP-1 greatly reduced Env localization in lipid rafts, and the property of Gag-independent gp41 localization in rafts was conserved among different strains. Analyses of mutants containing single deletions or substitutions in LLP-1 showed that the α-helical structure of the LLP-1 hydrophobic face has a more-critical role in Env-raft associations than that of the hydrophilic face. With the exception of a Pro substitution for Val-833, all Pro substitution and charge-inverting mutants showed wild-type virus-like one-cycle viral infectivity, replication kinetics, and Env incorporation into the virus. The intracellular localization and cell surface expression of mutants not localized in lipid rafts, such as the TM844, TM813, 829P, and 843P mutants, were apparently normal compared to those of wild-type Env. Cytoplasmic subdomain targeting analyses revealed that the sequence spanning LLP-3 and LLP-1 could target a cytoplasmic reporter protein to DRMs. Mutations of LLP-1 that affected Env association with lipid rafts also disrupted the DRM-targeting ability of the LLP-3/LLP-1 sequence. Our results clearly demonstrate that LLP motifs located in the C-terminal cytoplasmic tail of gp41 harbor Triton X-100-resistant raft association determinants.Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual in possessing a long cytoplasmic domain (∼150 amino acids) in their envelope (Env) transmembrane (TM) glycoprotein compared to those of other retroviruses (20 to 50 amino acids). The cytoplasmic domain of HIV-1 TM protein gp41, which encompasses residues 706 to 856, has multiple functions during the virus life cycle, including viral replication, infectivity, transmission, and cytopathogenicity. Truncations of the HIV-1 cytoplasmic domains may modulate cell-cell fusion properties of the Env protein, presumably due to alterations in the levels of cell surface Env expression and conformation of the Env ectodomain (23, 81). The cytoplasmic domain is characterized by the presence of three structurally conserved, amphipathic α-helical segments, located at residues 828 to 856, 770 to 795, and 786 to 813 and referred to as lentivirus lytic peptide 1 (LLP-1), LLP-2, and LLP-3, respectively, at its C terminus (Fig. ​(Fig.1A).1A). The LLP-1 and LLP-2 sequences were shown to be inserted into viral membranes by a photoinduced chemical reaction (73). These LLP motifs have been implicated in a variety of functions, such as cell surface expression (12), Env fusogenicity (30), and Env incorporation into a virus (47, 56), as well as Env protein stability (33) and multimerization (34).Open in a separate windowFIG. 1.(A) Schematic representation of the gp41 cytoplasmic domain and truncation mutants examined in this study. The cytoplasmic tail of gp41 contains a tyrosine-based endocytic YSPL signal located at residue 712, a hydrophilic region, a diaromatic YW motif, and three amphipathic α-helices, termed LLP-2, LLP-3, and LLP-1, at its C terminus. The amino acid sequence from residues 806 to 856 of the WT HXB2 Env is presented in single amino acid code, and the C-terminal dileucine motif is underlined in the sequence. Truncation mutants (TMs) generating stop codons immediately downstream of the indicated amino acids and their respective sequences are also shown. (B) pHXB2R3-based mutant proviruses used in this study. All mutants were generated by a PCR overlap cloning strategy, and the mutation sites are indicated. A dash or dot indicates that the residue in that position of the mutant provirus sequence is identical to or absent from that of the WT provirus sequence, respectively. The substituted amino acids in the mutant proviruses are also indicated.Gag and Env carry specific intracellular localization signals governing the site(s) of virus assembly/budding and release into the extracellular milieu. Env trafficking to the plasma membrane is regulated by the conserved C-terminal dileucine motif and the endocytic, membrane-proximal, tyrosine-based GY₇₁₂SPL signal in the cytoplasmic tail of gp41 (Fig. ​(Fig.1A)1A) and by their respective interactions with the clathrin adaptor proteins, AP1 and AP2 (4, 9, 21, 49, 65, 77). A diaromatic motif, Y₈₀₂W₈₀₃, was shown to bind to TIP47, a protein required for the retrograde transport of mannose-6-phosphate receptors from late endosomes to the trans-Golgi network, and this interaction was involved in the retrograde transport of Env to the trans-Golgi network (8). Alterations of these intracellular localization signals may affect viral infectivity, Env assembly into the virus, and viral replication (8, 20). Likewise, Gag also contains important sequences required for its trafficking to and assembly at the plasma membrane. The matrix (MA) protein, p17, contains a myristoyl group and a cluster of basic amino acids, while p6 contains a late domain which interacts with the components of the endosomal sorting complex required for transport (ESCRT) pathway to mediate Gag trafficking to the virion assembly/budding site (for reviews, see references 25, 45, 57, and 59). It is well documented that the specific interaction between the cytoplasmic domain of gp41 and the trimeric MA protein in infected cells facilitates recruitment of the Env into virus assembly/budding sites on target membranes (for reviews, see references 18, 24, and 46). TIP47 was demonstrated to act as an adaptor to bridge the gp41 cytoplasmic domain and Gag, which allows the physical encounter between Gag and Env, resulting in efficient Env incorporation into the virus during the viral assembly/budding process (39).Lipid rafts, also called detergent-resistant membranes (DRMs), are highly specialized membrane microdomains present in both the plasma and endosomal membranes of eukaryotic cells. These dynamic microdomains are characterized by their detergent insolubility, light density on a sucrose gradient, and enrichment of cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-linked proteins that are anchored in the membrane by their attached GPI moieties (1). HIV-1 utilizes lipid rafts to efficiently enter host cells (40, 74, 80) and selectively assembles and buds from lipid rafts on the surfaces of infected cells (27, 36, 48, 50, 54). Also, the HIV-1 Env protein was detected in lipid raft membranes (48, 54, 64). Lipid rafts are thought to facilitate Env-Gag interactions, to concentrate viral Env glycoproteins, and to promote multimerization of intracellular viral components (for a review, see reference 51). However, what governs Env transport to and localization in lipid rafts is a long-standing question.Although the mechanisms by which proteins associate with lipid rafts are not fully understood, determinants for targeting of signal proteins to DRMs have been identified. These include a GPI anchor (2, 61) and an N-terminal Met-Gly-Cys in which Gly is myristylated and Cys is palmitoylated (43, 71). The latter includes certain dually acylated heterotrimeric guanine nucleotide-binding protein (G protein) α subunits (44). In addition, acylation by palmitoylation also serves as a signal to target signaling molecules to lipid rafts (for reviews, see references 11 and 60). Some Env proteins of membrane-enveloped viruses are known to be associated with lipid rafts (35, 41, 54, 69, 79), and acylation of viral Env proteins, in particular, palmitoylation, is important for targeting these Env proteins to lipid rafts (for reviews, see references 58 and 70).It is generally believed that the association of HIV-1 Env with lipid rafts requires a palmitoylation signal(s) located in the cytoplasmic tail of gp41 (6, 64). Nevertheless, the two cytoplasmic palmitoylated Cys residues in the HXB2 strain Env protein are not conserved among HIV-1 isolates, and some isolates do not even contain cysteine residues in their cytoplasmic tail (32). In accordance with this notion, we previously demonstrated that the two cytoplasmic palmitoylated Cys residues in T-cell (T)- and macrophage (M)-tropic Env proteins do not play an obvious role in the virus life cycle, including Env''s association with lipid rafts (13), suggesting that other factors may substitute for cytoplasmic palmitoylation to promote Env localization in lipid rafts. Clapham''s group showed that mutations in MA or the cytoplasmic tail that prevent Env from incorporating into the virus and impair virus infectivity also interfere with Env''s association with lipid rafts (7), indicating that the Gag-Env interaction drives efficient Env association with lipid rafts, which in turn modulates Env budding and assembly onto viral particles. In contrast to their findings, we previously also noted that the Env protein of the HXB2 strain expressed without Gag is still located in lipid rafts (13), providing compelling evidence for the proposal that the Env per se contains sufficient information for its sequestration into lipid rafts.To further understand the nature of Env''s association with lipid rafts, in the present study we show that sequestering Env in Triton X-100-resistant lipid rafts is an intrinsic property of Env and is independent of Gag-Env interactions. Additionally, the LLP motifs, in particular the α-helical structure of the hydrophobic face of LLP-1, play a crucial role in Env''s localization in lipid rafts. Except for the 833P mutant of Env, which is unstable and degrades (33), all Pro-substituted mutants not located in lipid rafts exhibited wild-type (WT)-like phenotypes of intracellular localization, cell surface expression, incorporation into virions, and viral replication capacity. Importantly, the α-helix of the hydrophobic face of LLP-1 is also critical for the raft-targeting ability of the LLP-3/LLP-1 sequence. Our study depicts, for the first time, the critical role of the α-helix of the gp41 cytoplasmic domain in mediating Env''s association with and targeting to Triton X-100-resistant lipid rafts.     

Site-specific mutations in HIV-1 gp41 reveal a correlation between HIV-1-mediated bystander apoptosis and fusion/hemifusion

The loss of CD4(+) T cells in HIV-1 infections is hypothesized to be caused by apoptosis of bystander cells mediated by cell surface-expressed HIV-1 Env glycoprotein. However, the mechanism by which Env mediates this process remains controversial. Specifically, the role of HIV-1 gp120 binding to CD4 and CXCR4 versus the fusion process mediated by gp41 remains unresolved. Env-induced apoptosis in bystander cells has been shown to be gp41-dependent and correlates with the redistribution of membrane lipids between Env-expressing cells and target cells (hemifusion). Using a rational mutagenesis approach aimed at targeting Env function via the gp41 subunit, we examined the role of HIV gp41 in bystander apoptosis. A mutation in the fusion domain of gp41 (V513E) resulted in a fusion-defective Env that failed to induce apoptosis. A mutation in the gp41 N-terminal helix (G547D) reduced cell fusion capacity and apoptosis; conversely, an Env mutant with a deletion of the gp41 cytoplasmic tail (Ct Del) enhanced both cell-to-cell fusion and apoptosis. Most significantly, an Env mutant containing a substitution in the loop region of gp41 (D589L) mediated transfer of lipids (hemifusion) to bystander cells but was defective in cell-to-cell and to a lesser degree virus-to-cell fusion. This mutant was still able to induce apoptosis in bystander cells. Hence, we have provided the first direct evidence that gp41-mediated hemifusion is both required and sufficient for induction of apoptosis in bystander cells. These results may help to explain the mechanism of HIV-1 Env-induced T cell depletion.     

Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins.

The role of the cytoplasmic domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in virus replication was investigated. Deletion of residues 840 to 856 at the carboxyl terminus of gp41 reduced the efficiency of virus entry during an early step in the virus life cycle between CD4 binding and formation of the DNA provirus without affecting envelope glycoprotein synthesis, processing, or syncytium-forming ability. Deletion of residues amino terminal to residue 846 was associated with decreased stability of envelope glycoproteins made in COS-1 cells, but this phenotype was cell type dependent. The cytoplasmic domain of gp41 was not required for the incorporation of the HIV-1 envelope glycoproteins into virions. These results suggest that the carboxyl terminus of the gp41 cytoplasmic domain plays a role in HIV-1 entry other than receptor binding or membrane fusion. The cytoplasmic domain of gp41 also affects the stability of the envelope glycoprotein in some cell types.     

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.