Characterization of stable Chinese hamster ovary cells expressing wild-type, secreted, and glycosylphosphatidylinositol-anchored human immunodeficiency virus type 1 envelope glycoprotein.

We generated Chinese hamster ovary cell lines that stably express wild-type, secreted, and glycosylphosphatidylinositol (GPI)-anchored envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The cells expressing wild-type Env (WT cells) express both the precursor gp160 and the mature gp120/gp41 and readily form large syncytia when cocultivated with CD4+ human cells. The cells expressing secreted Env (SEC cells) release 140-kDa precursor and mature 120-kDa envelope glycoproteins into the supernatants. The cells expressing GPI-anchored Env (PI cells) express both 140-kDa precursor and mature gp120/gp41 envelope glycoproteins, which can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Both the secreted and PI-PLC-released envelope glycoproteins form oligomers that can be detected on nonreducing sodium dodecyl sulfate-polyacrylamide gels. In contrast to the WT cells, the SEC and PI cells do not form syncytia when cocultivated with CD4+ human cells. The availability of cells producing water-soluble oligomers of HIV-1 Env should facilitate studies of envelope glycoprotein structure and function. The WT cells, which readily induce syncytia with CD4+ cells, provide a convenient system for assessing potential fusion inhibitors and for studying the fusion mechanism of the HIV Env glycoprotein.     

Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4.

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.     

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.     

Proteolytic Processing of the Human Immunodeficiency Virus Envelope Glycoprotein Precursor Decreases Conformational Flexibility

The mature envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) virions is derived by proteolytic cleavage of a trimeric gp160 glycoprotein precursor. Remarkably, proteolytic processing of the HIV-1 Env precursor results in changes in Env antigenicity that resemble those associated with glutaraldehyde fixation. Apparently, proteolytic processing of the HIV-1 Env precursor decreases conformational flexibility of the Env trimeric complex, differentially affecting the integrity/accessibility of epitopes for neutralizing and nonneutralizing antibodies.     

Enzyme digests eliminate nonfunctional Env from HIV-1 particle surfaces, leaving native Env trimers intact and viral infectivity unaffected

HIV-1 viruses and virus-like particles (VLPs) bear nonnative "junk" forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer VLPs" may be useful neutralizing antibody immunogens.     

Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes.

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor glycoprotein, gp160, proceeds via the endoplasmic reticulum and Golgi complex and involves proteolytic processing of gp160 into the mature virion components, gp120 and gp41. We found that coexpression of gp160 and human CD4 in HeLa cells severely impaired gp120 production due to the formation of intracellular gp160-CD4 complexes. This CD4-mediated inhibition of gp160 processing was alleviated by coexpression of the HIV-1-encoded Vpu protein. The coexpression of Vpu and CD4 in the presence of gp160 resulted in increased degradation of CD4. Although the precise mechanism(s) responsible for the Vpu effect is presently unclear, our findings suggest that Vpu may destabilize intracellular gp160-CD4 complexes.     

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.     

Purification and characterization of the external envelope glycoprotein from two human immunodeficiency virus type 1 variants, HTLV-IIIB and HTLV-IIIRF.

External envelope glycoprotein from cell membranes and culture media of H9 cells infected with human immunodeficiency virus type 1 (HIV-1) isolate HTLV-IIIRF was isolated by immunoaffinity chromatography and compared with similar materials isolated from another variant, HTLV-IIIB. Envelope glycoprotein from IIIB and IIIRF appears to be identical, whether isolated from infected cell membranes or culture media. The molecular size of the IIIRF external envelope glycoprotein was 110 kilodaltons, whereas the relative size of IIIB gp120 was 123 kilodaltons. Amino-terminal sequence analysis of purified external envelope glycoprotein isolated from infected cell membranes or culture fluids revealed identical single sequences for the first 20 amino acids for each variant. The sequences obtained for IIIB gp120 were identical to those reported for the BH10 clone of the IIIB isolate, and the sequences determined for IIIRF gp110 matched the amino acid sequence predicted for the HAT3 clone of the Haitian HIV isolate. The amino-terminal sequences of external envelope glycoproteins isolated from either HIV-1 variant corresponded to the sequence starting at the proposed proteolytic cleavage site for the processing of the signal peptide of gp160. Immunization with external envelope glycoprotein isolated from either of the two HIV-1 variants yielded goat antibodies that primarily precipitated the homologous antigen. Sequential immunization of a single goat with gp120 and then gp110 resulted in the generation of antibodies that precipitated external envelope glycoprotein from both variants.     

Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates

Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

Inhibition of V3-specific cleavage of recombinant HIV-1 gp120 produced in Chinese hamster ovary cells

Specific proteolytic cleavage of the gp120 subunit of the HIV-1 envelope (Env) glycoprotein in the third variable domain (V3) has previously been reported to occur in several cell lines, including Chinese hamster ovary cells that have been used for production of Env-based HIV vaccine candidates. Here we report that this proteolytic activity on JRCSF gp120 is dependent on cell density, medium conditions, and supernatant concentration. The resulting cleaved polypeptides cannot be separated from intact gp120 by conventional or affinity chromatography under non-reducing conditions. Inhibitor studies reveal that Pefabloc and benzamidine, but not chymostatin, block gp120 cleavage in a dose-dependent fashion, suggesting the presence of a trypsin-like serine protease in CHO-K1 cells. The proteolytic activity is increased with certain types of cell culture growth media. A combination of serum-free OptiMEM media during expression and potent protease inhibitors post-expression can effectively prevent HIV gp120 degradation. The same strategy can be applied to the expression and purification of gp120 of other strains or other forms of envelope-based vaccine candidates containing V3 sequences.     

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.     

Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. Using a transient-expression system, we investigated the interaction of the HIV-1 envelope glycoprotein with CD4 during their movement through the intracellular membrane traffic. In singly transfected cells, the envelope glyprotein gp160 was synthesized, glycosylated, and localized predominantly in the endoplasmic reticulum. Only a minor fraction of gp160 was proteolytically cleaved, producing gp120 and gp41, and gp120 was secreted into the medium. On the other hand, the CD4 molecule, when expressed alone, was properly glycosylated and transported efficiently to the cell surface. However, when gp160 and CD4 were coexpressed in the same cell, the cell surface delivery of CD4 was greatly reduced. In coexpressing cells, CD4 formed a specific intracellular complex with gp160 as both proteins could be immunoprecipitated by antibodies against either the gp160 or CD4 (OKT4) but not by OKT4A, a blocking antibody against CD4. The specific gp160-CD4 complex was localized predominantly in the endoplasmic reticulum, and the CD4 in the complex did not acquire endoglycosidase H resistance. The present studies demonstrated that a specific intracellular interaction between gp160 and CD4 was responsible for the cell surface down-regulation of CD4 in cells expressing both the envelope glycoprotein of HIV-1 and its receptor, CD4.     

Human Ubc9 Is Involved in Intracellular HIV-1 Env Stability after Trafficking out of the Trans-Golgi Network in a Gag Dependent Manner

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH₂), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH₂ was uncovered by examining Env expression and maturation in treated cells. GPG-NH₂ treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH₂ negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH₂, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.The endoplasmic reticulum (ER) contains a number of molecular chaperones and folding factors that aid in the maturation of proteins that traverse the secretory pathway. This process is strictly monitored by the ER quality control system, which selects properly folded proteins for export to the Golgi (16) and targets misfolded proteins for destruction through the ER-associated protein degradation pathway (ERAD) (4, 28). Once an ER protein is selected as a substrate for ERAD, it is translocated from the ER lumen to the cytosol through an ER translocon. This retrotranslocation process is thought to be driven by either the cytosolic AAA-ATPase p97 (39) or the 19S proteasome cap (23). Upon entrance into the cytosol, the ERAD substrate is ubquitinated, and its glycans are removed by an N-glycanase to prepare it for proteasomal degradation (11, 28).Viral envelope glycoproteins utilize the host cell secretory pathway for their proper maturation and trafficking to the site of viral assembly. The human immunodeficiency virus type 1 (HIV-1) encodes the envelope glycoprotein (Env), which initiates HIV-1 infections by mediating attachment and fusion of the viral envelope with the host cell membrane (17). Therefore, infectious HIV-1 particle production relies on the ability of Env to pass the rigorous ER quality control system.Env is initially synthesized as a type I membrane precursor glycoprotein termed gp160, which is cotranslationally targeted to the ER by its 30-amino-acid N-terminal signal sequence (24). Within the ER, gp160 receives ∼30 N-linked glycans and is assisted in its maturation by the chaperones BiP, calnexin, and calreticulin as it undergoes extensive disulfide bond formations (15, 21, 31). Once gp160 has reached its native state with ten disulfide bonds and its signal sequence has been cleaved posttranslationally (21, 25), it assembles into trimers (26) and is exported to the Golgi. Within the Golgi, gp160 is cleaved by cellular endoproteases, yielding the transmembrane protein gp41 and the noncovalently associated surface protein gp120 (27). Thereafter, this complex is transported to the plasma membrane, where it is incorporated into the envelope of assembling HIV-1 particles.We have previously shown that a tripeptide amide corresponding to a conserved motif of the HIV-1 Env, glycyl-prolyl-glycine amide (GPG-NH₂), suppressed the replication of all 47 HIV-1 laboratory strains and clinical isolates examined with a 50% inhibitory concentration of ∼10 μM, a concentration that is 200- to 2,000-fold less than what affected cell growth or had other toxic effects on peripheral blood mononuclear cells (35). However, this suppression was not, as we had anticipated, due to interactions of the peptide with the early events of the HIV-1 replication cycle, such as attachment or entry (36). In the present study, we demonstrate that GPG-NH₂ reduced Env incorporation into HIV-1 particles during replication by targeting Env toward the ERAD pathway. The ability of GPG-NH₂ to target Env for degradation was dependent on the presence of functional proteasomes and required the full-length Env signal sequence. These findings illustrate that small molecules may be utilized therapeutically to specifically target unwanted pathogenic proteins for degradation by the existing cellular machinery.     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein.

The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms an inner coat directly underneath the lipid envelope of the virion. The outer surface of the lipid envelope surrounding the capsid is coated by the viral Env glycoproteins. We report here that the HIV-1 capsid-Env glycoprotein association is very sensitive to minor alterations in the MA protein. The results indicate that most of the MA domain of the Gag precursor, except for its carboxy terminus, is essential for this association. Viral particles produced by proviruses with small missense or deletion mutations in the region coding for the amino-terminal 100 amino acids of the MA protein lacked both the surface glycoprotein gp120 and the transmembrane glycoprotein gp41, indicating a defect at the level of Env glycoprotein incorporation. Alterations at the carboxy terminus of the MA domain had no significant effect on the levels of particle-associated Env glycoprotein or on virus replication. The presence of HIV-1 MA protein sequences was sufficient for the stable association of HIV-1 Env glycoprotein with hybrid particles that contain the capsid (CA) and nucleocapsid (NC) proteins of visna virus. The association of HIV-1 Env glycoprotein with the hybrid particles was dependent upon the presence of the HIV-1 MA protein domain, as HIV-1 Env glycoprotein was not efficiently recruited into virus particles when coexpressed with authentic visna virus Gag proteins.