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

The human immunodeficiency virus type 1 (HIV-1) envelope comprises a surface gp120 and a transmembrane gp41. The cytoplasmic domain of gp41 contains cysteine residues (C764 and C837) which are targets for palmitoylation and were reported to be required for envelope association with lipid rafts and assembly on budding virions (I. Rousso, M. B. Mixon, B. K. Chen, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:13523-13525, 2000). Several infectious HIV-1 clones contain envelopes that have no gp41 cytoplasmic cysteines. Since no other gp41 amino acid is a target for palmitoylation, these clones imply that palmitoylation is not essential for envelope trafficking and assembly. Here, we show that HIV-1 envelope mutants that lack gp41 cytoplasmic cysteines are excluded from light lipid rafts. Envelopes that contained residues with bulky hydrophobic side chains instead of cysteines retained their association with heavy rafts and were nearly fully functional for incorporation into virions and infectivity. Substitution of cysteines with alanines or serines eliminated raft association and more severely reduced envelope incorporation onto virions and their infectivity. Nevertheless, the A764/A837 mutant envelope retained nearly 40% infectivity compared to the wild type, even though this envelope was excluded from lipid rafts. Our results demonstrate that gp41 cytoplasmic cysteines that are targets for palmitoylation and are required for envelope trafficking to classical lipid rafts are not essential for HIV-1 replication.     

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.     

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.     

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.     

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.     

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.     

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.     

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains.

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms the outer protein shell directly underneath the lipid envelope of the virion. The MA protein has a key role in different aspects of virus assembly, including the incorporation of the HIV-1 Env protein complex, which contains a transmembrane glycoprotein with an unusually long cytoplasmic tail. In this study, we compared the abilities of HIV-1 MA mutants to incorporate Env protein complexes with long and short cytoplasmic tails. While the mutant particles failed to incorporate the authentic HIV-1 Env protein complex, they retained the ability to efficiently and functionally incorporate the amphotropic murine leukemia virus Env protein complex, which has a short cytoplasmic tail. Moreover, incorporation of the autologous Env protein complex could be restored by a second-site mutation that resulted in the truncation of the cytoplasmic tail of the HIV-1 transmembrane glycoprotein. Remarkably, the second-site mutation also restored the ability of MA mutants to replicate in MT-4 cells. These results imply that the long cytoplasmic tail of the transmembrane glycoprotein is responsible for the exclusion of the HIV-1 Env protein complex from MA mutant particles.     

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.     

Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity

Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag co-clustering and VS assembly in T-cell conjugates.     

CD4 raft association and signaling regulate molecular clustering at the immunological synapse site

T cell activation is associated with the partitioning of TCRs and other signaling proteins, forming an immunological synapse. This study demonstrates a novel function for the CD4 coreceptor in regulating molecular clustering at the immunological synapse site. We show using transgenic mouse and retroviral reconstitution studies that CD4 is required for TCR/protein kinase C (PKC) theta clustering. Specifically, we demonstrate that CD4 palmitoylation sequences are required for TCR/PKCtheta raft association and subsequent clustering, indicating a particular role for raft-associated CD4 molecules in regulating immune synapse organization. Although raft association of CD4 is necessary, it is not sufficient to mediate clustering, as cytoplasmic tail deletion mutants are able to localize to rafts, but are unable to mediate TCR/PKCtheta clustering, indicating an additional requirement for CD4 signaling. These studies suggest that CD4 coreceptor function is regulated not only through its known signaling function, but also by posttranslational lipid modifications which regulate localization of CD4 in lipid rafts.     

Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.     

Palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression

To investigate the association of the murine leukemia virus (MuLV) Env protein with lipid rafts, we compared wild-type and palmitoylation-deficient mutant Env proteins by using extraction with the mild detergent Triton X-100 (TX-100) followed by a sucrose gradient flotation assay. We found that the wild-type MuLV Env protein was resistant to ice-cold TX-100 treatment and floated to the top of the gradients. In contrast, we observed that the palmitoylation-deficient mutant Env protein was mostly soluble when extracted by ice-cold TX-100 and stayed at the bottom of the gradients. Both the wild-type and mutant Env proteins were found to be soluble when treated with methyl-beta-cyclodextrin before extraction with ice-cold TX-100 or when treated with ice-cold octyl-beta-glucoside instead of TX-100. These results indicate that the MuLV Env protein is associated with lipid rafts and that palmitoylation of the Env protein is critical for lipid raft association. Although the palmitoylation-deficient Env mutant was synthesized at a level similar to that of the wild-type Env, it was found to be expressed at reduced levels on the cell surface. We observed syncytium formation activity with both the wild-type and mutant Env proteins, indicating that palmitoylation or raft association is not required for MuLV viral fusion activity.     

Requirement for an intact T-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.     

Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.     

Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.     

Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck,and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling

By mutagenesis, we demonstrated that the palmitoylation of the membrane-proximal Cys(396) and Cys(399)of CD4, and the association of CD4 with Lck contribute to the enrichment of CD4 in lipid rafts. Ab cross-linking of CD4 induces an extensive membrane patching on the T cell surface, which is related to lipid raft aggregation. The lipid raft localization of CD4 is critical for CD4 to induce the aggregation of lipid rafts. The localization of CD4 in lipid rafts also correlates to the ability of CD4 to enhance receptor tyrosine phosphorylation. Thus, our data suggest that CD4-induced aggregation of lipid rafts may play an additional role in CD4 signaling besides its adhesion to MHC molecules and association with Lck.     

Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells

In this report, we describe a crucial role of lipid raft-colocalized receptors in the entry of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T cells. We show that biochemically isolated detergent-resistant fractions have characteristics of lipid rafts. Lipid raft integrity was required for productive HIV-1 entry as determined by (i) semiquantitative PCR analysis and (ii) single-cycle infectivity assay using HIV-1 expressing the luciferase reporter gene and pseudotyped with HIV-1 HXB2 envelope or vesicular stomatitis virus envelope glycoprotein (VSV-G). Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) relocalized raft-resident markers to a nonraft environment but did not significantly change the surface expression of HIV-1 receptors. MbetaCD treatment inhibited productive infection of HIV-1 by 95% as determined by luciferase activity in cells infected with HXB2 envelope-pseudotyped virus. In contrast, infection with VSV-G-pseudotyped virus, which enters the cells through an endocytic pathway, was not suppressed. Biochemical fractionation and confocal imaging of HIV-1 receptor distribution in live cells demonstrated that CD4, CCR5, and CXCR4 colocalized with raft-resident markers, ganglioside GM1, and glycosylphosphatidylinositol-anchored CD48. While confocal microscopy analysis revealed that HIV-1 receptors localized most likely to the same lipid microdomains, sucrose gradient analysis of the receptor localization showed that, in contrast to CD4 and CCR5, CXCR4 was associated preferentially with the nonraft membrane fraction. The binding of HIV-1 envelope gp120 to lipid rafts in the presence, but not in the absence, of cholesterol strongly supports our hypothesis that raft-colocalized receptors are directly involved in virus entry. Dramatic changes in lipid raft and HIV-1 receptor redistribution were observed upon binding of HIV-1 NL4-3 to PM1 T cells. Colocalization of CCR5 with GM1 and gp120 upon engagement of CD4 and CXCR4 by HIV-1 further supports our observation that HIV-1 receptors localize to the same lipid rafts in PM1 T cells.     

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the ΔYI, ΔIK, and ΔLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal α-helical heptad repeats, respectively, inhibited WT and ΔLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.