Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Glycosaminoglycans and protein disulfide isomerase-mediated reduction of HIV Env

Conformational changes within the human immunodeficiency virus-1 (HIV-1) surface glycoprotein gp120 result from binding to the lymphocyte surface receptors and trigger gp41-mediated virus/cell membrane fusion. The triggering of fusion requires cleavage of two of the nine disulfide bonds of gp120 by a cell-surface protein disulfide-isomerase (PDI). Soluble glycosaminoglycans such as heparin and heparan sulfate bind gp120 via V3 and, possibly, a CD4-induced domain. They exert anti-HIV activity by interfering with the HIV envelope glycoprotein ( Env)/cell-surface interaction. Env also binds cell-surface glycosaminoglycans. Here, using surface plasmon resonance, we observed an inverse relationship between heparin binding by gp120 and its thiol content. In vitro, and in conditions in which gp120 could bind CD4, heparin and heparan sulfate reduced PDI-mediated gp120 reduction by approximately 80%. Interaction of Env with the surface of lymphocytes treated using sodium chlorate, an inhibitor of glycosaminoglycan synthesis, led to gp120 reduction. We conclude that besides their capacity to block Env/cell interaction, soluble glycosaminoglycans can effect anti-HIV activity via interference with PDI- mediated gp120 reduction. In contrast, their presence at the cell surface is dispensable for Env reduction during the course of interaction with the lymphocyte surface. This work suggests that the reduction of exofacial proteins in various diseases can be inhibited by compounds targeting the substrates ( not by targeting PDI, as is usually done), and that glycosaminoglycans that primarily protect proteins by preserving them from proteolysis also have a role in preventing reduction.

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.

Significant redox insensitivity of the functions of the SARS-CoV spike glycoprotein - Comparison with HIV envelope

The capacity of the surface glycoproteins of enveloped viruses to mediate virus/cell binding and membrane fusion requires a proper thiol/disulfide balance. Chemical manipulation of their redox state using reducing agents or free sulfhydryl reagents affects virus/cell interaction. Conversely, natural thiol/disulfide rearrangements often occur during the cell interaction to trigger fusogenicity, hence the virus entry. We examined the relationship between the redox state of the 20 cysteine residues of the SARS-CoV (severe acute respiratory syndrome coronavirus) Spike glycoprotein S1 subdomain and its functional properties. Mature S1 exhibited similar to 4 unpaired cysteines, and chemically reduced S1 displaying up to similar to 6 additional unpaired cysteines still bound ACE2 and enabled fusion. In addition, virus/cell membrane fusion occurred in the presence of sulfhydryl-blocking reagents and oxidoreductase inhibitors. Thus, in contrast to various viruses including HIV (human immunodeficiency virus) examined in parallel, the functions of the SARS-CoV Spike glycoprotein exhibit a significant and surprising independence of redox state, which may contribute to the wide host range of the virus. These data suggest clues for molecularly engineering vaccine immunogens.

The chemokine receptor, CCR5

The chemokine receptor, CCR5, is a G protein coupled receptor responsible for some of the effects of the chemokines CCL3, CCL4 and CCL5. It is also one of the co-receptors for the entry of human immunodeficiency virus-1 (HIV-1) into cells. Regulation of CCR5 number on cells is, therefore, important for determining the infection rate by HIV-1. (C) 2003 Elsevier Ltd. All rights reserved.

An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.

NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-beta promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-beta induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-beta induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

Infection of human airway epithelium by human and avian strains of influenza a virus

We describe the characterization of influenza A virus infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). Sialic acid receptors for both human and avian viruses, alpha-2,6- and alpha-2,3-linked sialic acids, respectively, were detected on the HAE cell surface, and their distribution accurately reflected that in human tracheobronchial tissue. Nonciliated cells present a higher proportion of alpha-2,6-linked sialic acid, while ciliated cells possess both sialic acid linkages. Although we found that human influenza viruses infected both ciliated and nonciliated cell types in the first round of infection, recent human H3N2 viruses infected a higher proportion of nonciliated cells in HAE than a 1968 pandemic-era human virus, which infected proportionally more ciliated cells. In contrast, avian influenza viruses exclusively infected ciliated cells. Although a broad-range neuraminidase abolished infection of HAE by human parainfluenza virus type 3, this treatment did not significantly affect infection by influenza viruses. All human viruses replicated efficiently in HAE, leading to accumulation of nascent virus released from the apical surface between 6 and 24 h postinfection with a low multiplicity of infection. Avian influenza A viruses also infected HAE, but spread was limited compared to that of human viruses. The nonciliated cell tropism of recent human H3N2 viruses reflects a preference for the sialic acid linkages displayed on these cell types and suggests a drift in the receptor binding phenotype of the H3 hemagglutinin protein as it evolves in humans away from its avian virus precursor.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.