Cells expressing the human foamy virus (HFV) accessory Bet protein are resistant to productive HFV superinfection

Bet is a foamy virus (FV) accessory protein not required for virus replication. The function of Bet is not understood. We report on the generation of cell lines stably expressing the HFV Bet protein. In Bet+ cells, HFV replication was reduced by approximately 3-4 orders of magnitude compared with control cells. The HFV Bet-expressing cells only partially resisted infection by the distantly related feline FV (FFV). Pseudotyping experiments, using murine retroviral vectors with an HFV envelope, revealed that the resistance was not due to downregulation of the unknown HFV receptor. In transfection experiments, using proviral reporter gene constructs and infectious proviruses, no significant differences were detected between Bet+ and control cells. In infection experiments, HFV vectors expressing an indicator gene under control of the HFV promoters showed no activity in Bet+ cells. The results are best compatible with the hypothesis that the main block to productive superinfection of Bet+ cells occurs at an early stage of replication between virus entry and provirus establishment. We suggest that inhibition of provirus integration by Bet protein may serve a distinct function in the unique foamy virus replication cycle.     

Increased incorporation of chimeric human immunodeficiency virus type 1 gp120 proteins into Pr55gag virus-like particles by an Epstein-Barr virus gp220/350-derived transmembrane domain

Noninfectious Pr55gag virus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimeric env genes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein-Barr virus (EBV) major Env glycoprotein gp220/ 350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gag polyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5-10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gag virus-like particles.     

Mutational analysis of residues in the coiled-coil domain of human immunodeficiency virus type 1 transmembrane protein gp41

The envelope glycoprotein (Env) of human immunodeficiency virus mediates virus entry into cells by undergoing conformational changes that lead to fusion between viral and cellular membranes. A six-helix bundle in gp41, consisting of an interior trimeric coiled-coil core with three exterior helices packed in the grooves (core structure), has been proposed to be part of a fusion-active structure of Env (D. C. Chan, D. Fass, J. M. Berger, and P. S. Kim, Cell 89:263-273, 1997; W. Weissenhorn, A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley, Nature 387:426-430, 1997; and K. Tan, J. Liu, J. Wang, S. Shen, and M. Lu, Proc. Natl. Acad. Sci. USA 94:12303, 1997). We analyzed the effects of amino acid substitutions of arginine or glutamic acid in residues in the coiled-coil (heptad repeat) domain that line the interface between the helices in the gp41 core structure. We found that mutations of leucine to arginine or glutamic acid in position 556 and of alanine to arginine in position 558 resulted in undetectable levels of Env expression. Seven other mutations in six positions completely abolished fusion activity despite incorporation of the mutant Env into virions and normal gp160 processing. Single-residue substitutions of glutamic acid at position 570 or 577 resulted in the only viable mutants among the 16 mutants studied, although both viable mutants exhibited impaired fusion activity compared to that of the wild type. The glutamic acid 577 mutant was more sensitive than the wild type to inhibition by a gp41 coiled-coil peptide (DP-107) but not to that by another peptide corresponding to the C helix in the gp41 core structure (DP-178). These results provide insight into the gp41 fusion mechanism and suggest that the DP-107 peptide may inhibit fusion by binding to the homologous region in gp41, probably by forming a peptide-gp41 coiled-coil structure.     

Inhibition of CD4 translation mediated by human immunodeficiency virus type 1 envelope protein in a cell-free system

The human immunodeficiency virus type 1 (HIV-1) employs a number of complex strategies to interfere with the synthesis, stability, and subcellular localization of its specific cellular receptor CD4. To define better the mechanisms of inhibition of CD4 expression, we used a rabbit reticulocyte lysate in vitro system, in which cDNAs derived from HIV-1-infected cells were used to generate mRNA for the Tat, Vpu, and gp160 envelope proteins that were translated together with CD4-encoding mRNA. In the presence of microsomal membranes, we observed that cotranslation of Env mRNA resulted in a dose-dependent inhibition of CD4 translation. This effect was enhanced further when an mRNA-encoding Vpu in addition to Env mRNA was utilized. However, the activity of Vpu was mostly post-translational, since translation of Vpu alone, but not Env, was able to destabilize CD4 molecules presynthesized into microsomes. The Env-mediated inhibitory effect was specifically targeted at CD4 and did not affect the synthesis or stability of the CD8 molecule. Interestingly, mutated CD4 species, with a 20-fold lower affinity for HIV-1 Env than wild-type, were less sensitive to cotranslational inhibition. Our report identifies the envelope as the HIV-1 protein responsible for down-regulation of CD4 translation. We further propose a mechanism whereby direct interactions between gp160 and nascent CD4 molecules can cause interference with and premature termination of CD4 protein elongation.     

Recombinant human immunodeficiency Pr55gag virus-like particles presenting chimeric envelope glycoproteins induce cytotoxic T-cells and neutralizing antibodies

Very recently, we demonstrated that the replacement of the human immunodeficiency virus type-1 (HIV-1) gp41 transmembrane protein by an Epstein-Barr virus gp220/350-derived membrane anchor resulted in the incorporation of chimeric envelope (Env) oligomers into Pr55gag virus-like particles (VLPs), exceeding that of wild-type gp160 by a factor of 10. In this study, we examined the immunostimulatory properties of Pr55gag VLPs to both (i) chimeric HIV-1 gp120 external envelope proteins and (ii) full-length gp160 presented on the outer surface of the particles. Immunization studies carried out with VLPs presenting different derivatives of the chimeric and wild-type Env proteins elicited a consistent anti-Pr55gag as well as anti-Env antibody response in complete absence of additional adjuvants. In both cases, the immune sera exhibited an in vitro neutralizing activity against homologous HIV-1 infection in MT4 cells. Noteworthy, these VLPs were also capable of inducing a strong CD8+ cytotoxic T-cell (CTL) response in immunized BALB/c mice that was directed toward a known CTL epitope in the third variable domain V3 of the gp120 external glycoprotein. However, the induction of V3-loop-specific CTLs critically depended on the amounts of Env proteins that were presented by the Pr55gag VLPs. Moreover, the CD8+ CTL response was not significantly altered by adsorbing the VLPs to alum or by repeated booster immunizations. These results illustrate that Pr55gag VLPs provide a safe and effective means of enhancing neutralizing humoral responses to particle-entrapped gp120 proteins and are also capable of delivering these proteins to the MHC class I antigen processing and presentation pathway. Therefore, antigenically expanded Pr55gag VLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired.     

Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences

Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization.     

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes

We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.     

High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.     

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.     

Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS3 generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic alpha-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.     

Cooperative effects of the human immunodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells

Insertion of T-cell line-tropic V3 and V4 loops from the HXB2 strain into the macrophage-tropic YU-2 envelope resulted in a virus with delayed infectivity for HUT78 and Jurkat cells compared with HXB2. Sequence analysis of viral DNA derived from long-term cultures of Jurkat cells revealed a specific mutation that changed a highly conserved Asn residue in the V1 loop of Env to an Asp residue (N-136-->D). Introduction of this mutation into clones containing a T-cell line-tropic V3 loop, either with or without a T-cell line-tropic V4 loop, resulted in viruses that replicated to high levels in Jurkat cells and peripheral blood lymphocytes. The Env proteins from these constructs were expressed with the vaccinia virus/T7 hybrid system and were found to be translated, processed, and cleaved and to bind to soluble CD4 similar to the wild-type HXB2 and YU-2 Env proteins. Env-mediated fusion with HeLa T4+ cells, however, was regulated by both the altered V1 loop and T-cell line-tropic V3 loop. These results suggest that subsequent to the initial gp120-CD4 binding event, a functional interaction can occur between the altered V1 loop and T-cell line-tropic V3 loop that results in infection of Jurkat cells and peripheral blood lymphocytes.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.     

DNA vaccination using expression vectors carrying FIV structural genes induces immune response against feline immunodeficiency virus

Following inactivated virus vaccination trials, the surface glycoprotein gp120 of the feline immunodeficiency virus (FIV) was considered as one of the determinants for protection. However, several vaccination trials using recombinant Env protein or some peptides failed to induce protection. To understand the role of the gp120 protein in vivo, we vaccinated cats with naked DNA coding for FIV structural proteins gp120 and p10. We analyzed the ability of these vaccinations to induce immune protection and to influence the onset of infection. Injection in cat muscles of expression vectors coding for the FIV gp120 protein induced a humoral response. Cats immunized twice with the gp120 gene showed different patterns after challenge. Two cats were, like the control cats, infected from the second week after infection onwards. The two others maintained a low proviral load with no modification of their antibody pattern. The immune response induced by gp120 DNA injection could control the level of viral replication. This protective-like immune response was not correlated to the humoral response. All the cats immunized with the gp120 gene followed by the p10 gene were infected, like the control cats, from the second week but they developed a complete humoral response against viral proteins after challenge. Furthermore, they showed a sudden but transient drop of the proviral load at 4 weeks after infection. Under these conditions, one injection of the p10 gene after one injection of the gp120 gene was not sufficient to stimulate protection. On the contrary, after a period, it seems to facilitate virus replication.     

Glycoprotein 330, a member of the low density lipoprotein receptor family, binds lipoprotein lipase in vitro

Glycoprotein 330 (gp330), a cell-surface protein that is localized in clathrin-coated pits, is structurally related to both the low density lipoprotein receptor (LDLR) and the LDLR-related protein/alpha 2-macroglobulin receptor (LRP). We recently demonstrated that gp330 and LRP may be functionally related as well; both bind the 39-kDa polypeptide referred to as receptor-associated protein (Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166). In this report, we tested several other LRP ligands for their ability to interact with human and rat gp330 in vitro. Gp330 did not exhibit detectable binding to the LRP ligands, alpha 2-macroglobulin protease complex or Pseudomonas aeruginosa exotoxin A. However, we found that gp330 (purified from human or rat) bound the lipolytic enzyme lipoprotein lipase (LPL) with high affinity (Kd = 6.1 and 2.7 nM, respectively). The binding was saturable, divalent cation dependent, and inhibited by heparin or receptor-associated protein. Because LRP has also been shown to bind LPL, the present findings further extend the functional similarities between gp330 and LRP. By analogy to the postulated role of the LRP-LPL interaction in facilitating hepatic clearance of LPL-associated lipoproteins from the blood (Beisiegel, U., Weber, W., and Bengtsson-Olivercrona, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8342-8346; Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767), we speculate that the gp330-LPL interaction described herein may contribute to the uptake of LPL-associated lipoproteins in tissues expressing gp330. Consistent with this possibility, we found that LPL promoted in vitro binding of 125I-lipoproteins to gp330.     

The course of childhood bronchiectasis: a case report and considerations of hospital use

Using an inhibitory synthetic peptide (DP-178) from HIV-1 gp41, we have trapped HIV-1 envelope glycoprotein (Env) undergoing conformational changes during virus entry. Our data show that DP-178 binds gp41 and inhibits Env-mediated membrane fusion after gp120 interacts with cellular receptors, indicating that conformational changes involving the coiled coil domain of gp41 are required for entry. Capture of this fusion-active conformation of Env provides insights into the early events leading to Env-mediated membrane fusion.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.