Artificial viral envelopes containing recombinant human immunodeficiency virus (HIV) gp160.

An artificial viral envelope was constructed, resembling the human immunodeficiency virus (HIV) envelope with respect to ultrastructure, size, phospholipid profile and lipid:cholesterol ratio. Recombinant HIV surface protein gp160 was anchored in the outer surface of the envelope membrane using a double detergent dialysis. The envelopes remained physically stable for several months. Immunolabeling with anti-gp160/41 monoclonal antibody revealed surface insertion and availability of gp160 for binding. Cell fusion and cytosolic transfer of the encapsulated fluorescent marker FITC-dextran was demonstrated. Flow cytometry indicated more efficient transfer of the fluorescent marker to cells which were approximately 60% CD4+ (REX-1B), relative to cells which were only approximately 18% CD4+ (KG-1). However, plain lipid envelopes without gp160 fused very efficiently with both cell types, indicating their potential usefulness as "fusogenic liposomes". Complete artificial viral envelopes may serve as subunit vaccines, and receptor-targeted delivery systems for drugs, toxins and genetic constructs.     

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.     

Synthesis and processing of human immunodeficiency virus type 1 envelope proteins encoded by a recombinant human adenovirus.

A recombinant adenovirus was constructed by inserting the human immunodeficiency virus type 1 (HIV-1) envelope gene downstream from the early region 3 (E3) promoter of adenovirus type 5 (Ad5), replacing the coding sequences of E3. The recombinant virus replicated as efficiently as the parent virus in all cell lines tested. Human cells infected with the recombinant virus synthesized the HIV-1 envelope precursor gp160, which was efficiently processed to the envelope glycoproteins gp120 and gp41. A human T-lymphoblast line (Molt-4) infected with the recombinant virus expressed HIV-1 envelope glycoproteins on the cell surface, leading to syncytium formation. The envelope gene was expressed from the E3 promoter at early times after infection and at late times from the major late promoter. When cotton rats were infected with the recombinant virus, antibodies against the HIV-1 envelope glycoproteins could be expressed in an immunoreactive form by the recombinant adenovirus, further illustrating the usefulness of adenoviruses as expression vectors.     

CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor.

We analyzed coexpression of the human immunodeficiency virus type 1 glycoprotein precursor, gp160, and its cellular receptor CD4 in HeLa cells to determine whether the two molecules can interact prior to transport to the cell surface. Results of studies employing coprecipitation, analysis of oligosaccharide processing, and immunocytochemistry showed that newly synthesized CD4 and gp160 form a complex prior to transport from the endoplasmic reticulum (ER). CD4 expressed by itself was transported efficiently from the ER to the cell surface, but the complex of CD4 and gp160 was retained in the ER. This retention of CD4 within the ER is probably a consequence of the very inefficient transport of gp160 itself (R. L. Willey, J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner, Proc. Natl. Acad. Sci. USA 85:9580-9584, 1988). Retention of CD4 in the ER by gp160 may partially explain the down regulation of CD4 in human immunodeficiency virus type 1-infected T cells. Inhibition of CD4 transport appears to be a consequence of the interaction of two membrane-bound molecules, because a complex of CD4 and gp120 (the soluble extracellular domain of gp160) was transported rapidly and efficiently from the ER.     

Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.     

Tumor necrosis factor-alpha inhibits the competence signal delivered by HIV to normal B cells

Polyclonal B cell activation is commonly observed in HIV-infected patients. The coordinate delivery of a number of signals is required for B cell response. This work was designed to better define the role of HIV in the first steps of normal human B cells activation. We show that the infectious virus or recombinant envelope proteins can render B cells responsive to the growth-promoting effect of several T cell-derived IL, IL-2, IL-4, and low m.w. (12-kDa) BCGF. HIV acts in the absence of monocytes and on different populations of B cells. The competence signal can be provided by recombinant gp160 envelope protein. CD4 molecule is not involved in the interaction of HIV with B cells. In addition, we demonstrate that tumor necrosis factor alpha has no promoting activity when B cells are preactivated by HIV and it can suppress the response of HIV-preactivated B cells to IL-2, IL-4, and 12-kDa BCGF. Thus, the HIV envelope can deliver an early signal to normal B cells and modulate B cell response to physiologic signals. The possible relevance of this phenomenon to the immune defect observed in HIV patients is discussed.     

Construction and use of a human immunodeficiency virus vector for analysis of virus infectivity.

We constructed a recombinant human immunodeficiency virus (HIV) vector to facilitate studies of virus infectivity. A drug resistance gene was inserted into a gp160- HIV proviral genome such that it could be packaged into HIV virions. The HIV genome was rendered replication defective by deletion of sequences encoding gp160 and insertion of a gpt gene with a simian virus 40 promoter at the deletion site. Cotransfection of the envelope-deficient genome with a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. The drug resistance gene was transmitted and expressed upon infection of susceptible cells, enabling their selection in mycophenolic acid. This system provides a quantitative measure of HIV infection, since each successful infection event leads to the growth of a drug-resistant colony. The HIV-gpt virus produced was tropic for CD4+ human cells and was blocked by soluble CD4. In the absence of gp160, noninfectious HIV particles were efficiently produced by cells transfected with the HIV-gpt genome. These particles packaged HIV genomic RNA and migrated to the same density as gp160-containing virions in a sucrose gradient. This demonstrates that HIV virion formation is not dependent on the presence of a viral envelope glycoprotein. Expression of a murine leukemia virus amphotropic envelope gene in cells transfected with HIV-gpt resulted in the production of virus capable of infecting both human and murine cells. These results indicate that HIV can incorporate envelope glycoproteins other than gp160 onto particles and that this can lead to altered host range. Like HIV type 1 and vesicular stomatitis virus(HIV) pseudotypes, gp-160+ HIV-gpt did not infect murine NIH 3T3 cells that bear human CD4, confirming that these cells are blocked at an early stage of HIV infection.     

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein.

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.     

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.     

Human immunodeficiency virus type 1 Vpu has a CD4- and an envelope glycoprotein-independent function.

Vpu is a 16-kDa membrane-associated phosphoprotein that is expressed from the same, singly spliced message as the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor, gp160. Previous studies suggest that Vpu functions in the late stages of viral replication, possibly in virus egression from the cell. Recently, it has been demonstrated that Vpu functions to allow gp160 to be more efficiently processed by disrupting CD4-gp160 complexes generated by transfection of HeLa cells. We show here that the lack of expression of intact Vpu results in a 90% reduction in infectious virus produced over a single round of replication from HeLa cells in the absence of CD4 expression. This reduction persists when HIV-1 particles are pseudotyped with the HIV-2 or amphotropic murine leukemia virus envelope glycoprotein. Pulse-chase analysis of HIV-1 capsid protein (p24) in the absence of CD4 and envelope glycoprotein demonstrates that the rate of virus release is reduced when Vpu is not expressed. Our findings indicate that Vpu has a function involving particle release not dependent on CD4 or envelope glycoprotein expression.     

The role of envelope glycoprotein processing in murine leukemia virus infection.

The murine leukemia virus envelope protein is synthesized as a precursor molecule, Pr85env, which is proteolytically cleaved at an arginine residue to produce two mature envelope proteins, gp70 and p15(E). The results presented here indicate that mutation to lysine of the arginine found at the envelope precursor cleavage site results in a precursor which is cleaved with an efficiency at least 10-fold lower than the efficiency with which the wild-type protein is cleaved. This mutation has been used to investigate the requirement for envelope protein processing in various aspects of retroviral infection. Viruses produced by cells transfected with mutant proviral clones are approximately 10-fold less infectious than wild-type viruses. Mutant viruses are incapable of inducing XC cell syncytium formation and are 100-fold less efficient than wild-type viruses at rendering cells resistant to superinfection. Envelope glycoproteins bearing the lysine mutation are found in reduced amounts on the surface of infected cells, and as a result mutant virions contain significantly less envelope protein than do wild-type virions. The phenotypic effects of the processing mutation described here are most likely the result of this paucity of envelope glycoproteins in virions carrying the mutation.     

The disulfide loop of gp41 is critical to the furin recognition site of HIV gp160

The importance of the HIV gp41 conserved disulfide loop to envelope function has been examined by mutational and functional analyses. Based on a luciferase-reporter entry assay, mutants gp41-CC/AA (C598A/C604A) and gp41-Delta (deletion of residues 596-606) result in a nonfunctional envelope protein. Western blot analysis shows both mutants to be properly expressed but not processed to form gp120 and gp41, which explains their nonfunctionality. The presence of mutant gp160 on the cell surface, as well as their ability to bind to sCD4, suggests that the mutations have disrupted processing at the furin recognition site encoded within the gp120 conserved domain 5, without resulting in an overall misfolding of the protein. With respect to the furin recognition site, the mutations are sequentially distant, which implies that the gp41 disulfide loop is interacting with gp120 C5 in gp160. In addition, we have modeled the gp120-gp41 interaction in unprocessed precursor gp160 using structural data available for gp120 and gp41 domains in isolation, supplemented by mutagenesis data. We suggest that the mutations have altered the interaction between gp120 C5 and the gp41 disulfide loop, resulting in decreased accessibility of the furin recognition site and implying that the interaction between the gp120 C5 and gp41 loop is a conformational requirement for gp160 processing. The sensitivity of this interaction could be exploited in future antivirals designed to disrupt HIV pathogenesis by disrupting gp160 processing.     

Effects of deletion of the cytoplasmic domain upon surface expression and membrane stability of a viral envelope glycoprotein

The envelope protein (gp52) of Friend spleen focus-forming virus (F-SFFV) is defective in its intracellular transport and accumulates in the rough endoplasmic reticulum of F-SFFV-infected cells. This defect in transport has been attributed to the lack of a cytoplasmic domain, and possible loss of signals required for transport to the cell surface. The mature form of gp52, designated gp65, is also reported to be secreted from SFFV-infected cells. To determine the specific changes in the envelope protein which may lead to its lack of transport and to its lack of stability in associating with membranes, the 3' end of the F-SFFV envelope gene, which encodes the transmembrane domain, was inserted in place of the normal 3' end of the Friend murine leukemia virus genome. This chimeric envelope gene was expressed using the vaccinia virus expression system. The chimeric gp70/p15E glycoprotein molecule lacks the cytoplasmic tail residues and as a consequence is about 3300 daltons smaller. The chimeric PrEnv molecule was found to be cleaved efficiently as indicated by pulse-chase experiments. Immunofluorescence studies demonstrate that the chimeric molecule is efficiently transported to the surface of cells, unlike the SFFV gp52 glycoprotein. The chimeric molecule was found to be unstable in its membrane association and is released into the culture medium. These results indicate that the changes in the membrane spanning region and the lack of a cytoplasmic tail do not determine the defective transport of gp52, but may determine the stability of its association with membranes.     

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.     

Improved antigenicity of the HIV env protein by cleavage site removal

The HIV env glycoprotein mediates virus infection and cell fusion through an interaction with the CD4 molecule present at the surface of T4+ lymphocytes. Although env presents a major antigenic target, vaccinia recombinants expressing env elicit low titres of anti-env antibody (Kieny et al., Bio/Technology, 4, 790-795, 1986). To delimit the functional domains of env and to improve the immunogenicity of the vaccinia recombinants we constructed variants expressing env proteins in which the site permitting cleavage of the gp160 precursor to yield gp120 and gp41 was removed, the gp120 and gp41 moieties separated or in which the signal sequence and hydrophobic domains were replaced by equivalents from rabies virus G. Analysis of variants revealed that the gp120 moiety is alone capable of interacting with CD4 and of provoking aggregation of T4+ lymphocytes, whereas cell-associated gp41 liberated by gp160 cleavage was essential for cell fusion. The identity of the signal and transmembrane zones however appeared unimportant. Although removal of the consensus sequence permitting cleavage of gp160 prevented syncytium formation but not aggregation of T4+ lymphocytes, significant cleavage continued to take place. Removal of a second potential cleavage site blocked gp160 cleavage. The live viruses were examined for immunogenicity: recombinant 1139 which lacks both putative cleavage sites was found to elicit a 10-fold higher antibody response in experimental animals than the parental recombinant.     

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

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.