Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.     

Processing of the envelope glycoprotein gp160 in immunotoxin-resistant cell lines chronically infected with human immunodeficiency virus type 1.

We describe the isolation and characterization of variant cell lines which are chronically infected with the human immunodeficiency virus (HIV) and resistant to the action of immunotoxins directed against the HIV envelope protein. These variants all produce normal levels of HIV proteins, budding virions, and the envelope protein precursor gp160. Two of the variants, 10E and 11E, contain a mutation within the env gene which results in the production of a truncated precursor and altered processing and transport of the protein to the cell surface. Variants B9 and G4 are defective in gp160 cleavage and do not efficiently transport the envelope protein to the cell surface. There are no mutations in the expressed viruses of B9 and G4. These cell lines express higher levels of CD4 protein and mRNA than H9/NL4-3. Thus, 10E, 11E, B9, and G4 have escaped immunotoxin action by downmodulating the envelope protein from their cell surfaces. None of these variants produce infectious HIV. Two other immunotoxin-resistant variants, E9-3 and 41-17, produce normal levels of gp160, efficiently transport the cleaved and processed subunits to the cell surface, and secrete infectious HIV. These studies identify alterations in gp160 processing that underscore the importance of the relationship between HIV and the cell that it infects.     

Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection

Correct endoproteolytic maturation of gp160 is essential for the infectivity of human immunodeficiency virus type 1. This processing of human immunodeficiency virus-1 envelope protein, gp160, into gp120 and gp41 has been attributed to the activity of the cellular subtilisin-like proprotein convertase furin. The prototypic furin recognition cleavage site is Arg-X-Arg/Lys-Arg. Arg-Arg-Arg-Arg-Arg-Arg or longer iterations of polyarginine have been shown to be competitive inhibitors of substrate cleavage by furin. Here, we tested polyarginine for inhibition of productive human immunodeficiency virus-1-infection in T-cell lines, primary peripheral blood mononuclear cells, and macrophages. We found that polyarginine inhibited significantly human immunodeficiency virus-1 replication at concentrations that were benign to cell cultures ex vivo and mice in vivo. Using a fluorogenic assay, we demonstrated that polyarginine potently inhibited substrate-specific proteolytic cleavage by furin. Moreover, we verified that authentic processing of human immunodeficiency virus-1 gp160 synthesized in human cells from an infectious human immunodeficiency virus-1 (HIV-1) molecular clone was effectively blocked by polyarginine. Taken together, our data support that inhibitors of proteolytic processing of gp160 may be useful for combating human immunodeficiency virus-1 and that polyarginine represents a lead example of such inhibitors.     

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.     

Theoretical and functional analysis of the SIV fusion peptide.

The fusion domain of simian immunodeficiency virus (SIV) envelope glycoproteins is a hydrophobic region located at the amino-terminal extremity of the transmembrane protein (gp32). Assuming an alpha helical structure for the SIV fusogenic domain of gp32 in a lipid environment, theoretical studies have predicted that the fusion peptide would insert obliquely in the lipid bilayer. This oblique insertion could be an initial step of the fusion process by disorganizing locally the structure of the lipid bilayer. We have tested this hypothesis by selectively mutagenizing the SIV gp160 expressed via a vaccinia virus vector, to alter the theoretical angle of insertion of the fusion peptide. The fusogenic activity of the wild-type and mutant glycoproteins was tested after infection of T4 lymphocytic cell lines by the recombinant vaccinia virus, and measure of syncytia formation. Mutations that modified the oblique orientation reduced the fusogenic activity. In contrast, mutations that conserve the oblique orientation did not alter the fusogenic properties. Our results support the hypothesis that oblique orientation is important for fusogenic activity.     

The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells.

Proteolytic activation of the precursor envelope glycoproteins gp160 of human immunodeficiency virus type 1 (HIV-1) and gp140 of HIV-2, a prerequisite for viral infection, results in the formation of gp120/gp41 and gp125/gp36, respectively. Cleavage is mediated by cellular proteases. Furin, a member of the eukaryotic subtilisin family, has been shown to be an activating protease for HIV. Here, we compared the presence of furin and other mammalian subtilisins in lymphatic cells and tissues. Northern blot analyses revealed that furin and the recently discovered protease LPC/PC7 were the only subtilisin-like enzymes transcribed in such cells. Furin was identified as an enzymatically active endoprotease present in different lymphocytic, as well as monocytic, cell lines. When expressed from vaccinia virus vectors, the proprotein convertases were correctly processed, transported, and secreted into the media and enzymatically active. Coexpression of different subtilisins with the HIV envelope precursors revealed that furin and LPC/PC7 are able to cleave HIV-1 gp160. Moreover, both enzymes proteolytically processed the envelope precursor of HIV-2. gp140 was also cleaved to some extent by PC1, which is not, however, present in lymphatic cells. Furin- and LPC/PC7-catalyzed cleavage of HIV-1 gp160 resulted in biologically active envelope protein. In conclusion, among the known members of the subtilisin family, only furin and LPC/PC7 fulfill the requirements of a protease responsible for in vivo activation of HIV envelope glycoproteins.     

Leucine zipper domain of HIV-1 gp41 interacted specifically with alpha-catenin

Interactions between viral and cellular proteins could explain the molecular mechanisms behind the viral life cycle of HIV-1. The envelope protein gp41 of HIV-1 specifically interacted with alpha-catenin, not with beta-catenin. This interaction was shown by in vitro protein assay and in vivo transfected cell systems. Microinjection of the DNA expressing HIV-1 gp160 and alpha-catenin, into the HeLa cell, resulted in the colocalization of gp41 and alpha-catenin. Interestingly the noncleavable mutant of gp160 and alpha-catenin were found to be colocalized in the cell membrane. Mapping of the interaction sites between these two proteins revealed that the leucine zipper-like structure, located between the first and second alpha-helix domains from the carboxy terminus of HIV-1 gp41, interacted strongly with the carboxy terminus of alpha-catenin.     

Characterization of murine-specific leukemia virus receptor from L cells.

The host cell receptor for Moloney murine leukemia virus was solubilized from murine L-cell membranes and characterized. In initial studies designed to identify a receptor-rich cell line, different mouse cells were screened for binding to Moloney gp70, the viral envelope glycoprotein which determines host cell-binding specificity. gp70 binding to murine L cells was specific and saturable, with an apparent affinity constant (Ka) of 4 X 10(8) M-1, and the number of receptors per cell (6 X 10(5)) was similar to that of other mouse fibroblast cell lines. Characterization of the gp70 receptor with regard to extraction by detergents, protease sensitivity, and heat denaturation suggests that the receptor is an intrinsic membrane protein. Upon extraction of L-cell membranes with 0.2% deoxycholic acid and precipitation with acetone, specific and saturable binding of gp70 could be detected. The solubilized gp70-binding component was eluted upon gel filtration on Sephacryl S-300 into a species with an approximate molecular weight of 110,000.     

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.     

A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1.

Furin, a subtilisin-like mammalian endoprotease, is thought to be responsible for the processing of many proprotein precursors of cellular and viral origin, including gp160 of human immunodeficiency virus type 1, which share the consensus processing site motif, Arg-X-Lys/Arg-Arg, for protease recognition (for reviews, see P. J. Barr, Cell 66:1-3, 1991, and Y. Nagai, Trends Microbiol. 1:81-87, 1993). To confirm and extend the concept that gp160 is processed by furin, we used here a cell line, LoVo, which was recently demonstrated to be furin defective. Unexpectedly, LoVo cells were found to process gp160 as efficiently as normal cell lines do, hence being able to fuse with CD4-expressing HeLa cells and to produce fully infectious virions. On the other hand, the same cell line was almost totally incapable of processing Newcastle disease virus fusion glycoprotein with a similar oligobasic cleavage recognition motif, providing a strong case for furin-mediated processing. Our present study thus raises a further need to search for and identify the proteinases involved in human immunodeficiency virus type 1 gp160 processing rather than supporting the notion that furin is responsible.     

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.     

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.     

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.     

Interactions between HIV-1 gp41 core and detergents and their implications for membrane fusion

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.     

Glycoprotein encoded by the Friend spleen focus-forming virus.

The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtitier test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Localization of a putative cell adhesion molecule (gp110) in Wistar and Fischer rat tissues

A plasma membrane glycoprotein (gp110) involved in cellular adhesion was studied in Wistar and Fischer rats. For quantitative analysis of the gp110 molecule a sandwich-ELISA was used. High quantities of gp110 were found especially in the liver, small intestine, submandibular gland and lung. The distribution and localization of the gp110 were investigated by immunohistochemistry utilizing soluble complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase antibodies. Immunoreactivity was present in plasma membranes of vascular endothelial cells of some organs. Furthermore, immunostaining also occurred in plasma membranes of lymphocytes, exocrine gland cells, excretory duct cells, hepatocytes, epithelial cells of the small intestine, kidney and vesicular gland and in the cytoplasm of renal connecting and collecting duct cells. The localization of gp110 in the luminal domain of the plasma membrane at many sites suggests that this glycoprotein is also involved in processes distinct from cell adhesion.     

Differential targeting of an epithelial plasma membrane glycoprotein in polarized Madin-Darby canine kidney cells

Using monoclonal antibodies directed against the plasma membrane of Madin-Darby canine kidney (MDCK) cells, we demonstrated previously that a glycoprotein with an Mr = 23,000 (gp23) had a non-polarized cell surface distribution and was observed on both the apical and basolateral membranes (Ojakian, G. K., Romain, R. E., and Herz, R. E. (1987) Am. J. Physiol. 253, C433-C443). However, in parallel studies on MDCK clonal lines (D11, D18) with high transepithelial electrical resistances and in kidney cells in vivo it was determined that gp23 had a polarized cell surface distribution, being localized only to the basolateral membrane. The cell surface distribution of other glycoproteins was identical in both MDCK and MDCK clonal lines, indicating that MDCK cells were not deficient in the ability to properly sort membrane glycoproteins. Metabolic labeling with radioactive substrates followed by immunopurification and gel electrophoresis demonstrated that gp23 from both MDCK and MDCK clone D11 had many biochemical similarities including electrophoretic mobility, glycosylation, and palmitate incorporation. However, proteolytic digestion of gp23 from MDCK and clone D11 cells produced unique peptide maps suggesting that these closely related glycoproteins may have different primary sequences. In this report, we present evidence that the differential targeting of gp23 may be due to differences between the primary sequences of the basolateral and non-targeted proteins. The possibility that the observed differences in gp23 targeting are due to the presence of a basolateral recognition signal in gp23 from clone D11 cells is discussed.