Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity.

Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.

BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

Biochemical indication for myristoylation-dependent conformational changes in HIV-1 Nef.

The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Disassembly of human immunodeficiency virus type 1 cores in vitro reveals association of Nef with the subviral ribonucleoprotein complex.

The human immunodeficiency virus type 1 (HIV-1) virulence factor Nef enhances viral infectivity in single-cycle infection assays and accelerates HIV-1 replication in vitro. It has been reported that the effects of Nef are mediated early after viral entry and before the completion of reverse transcription, as viral DNA synthesis is strongly attenuated during infection by Nef-defective virions. Our previous work has demonstrated that Nef is associated with mature HIV-1 cores, implicating Nef in the regulation of HIV-1 core stability. Here we report a comparative analysis of HIV-1 cores isolated from wild-type and Nef-defective particles. We observed no effect of Nef on HIV-1 core structure or stability; however, Nef cosedimented with a subviral ribonucleoprotein complex following dissociation of CA. These results indicate that Nef interacts tightly with an internal component of the HIV-1 core. They further suggest that virion-associated Nef may facilitate an early step in HIV-1 infection following dissociation of the viral capsid in the target cell.

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family.

Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention.

The association of Nef with a cellular serine/threonine kinase and its enhancement of infectivity are viral isolate dependent.

The nef genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) encode a 27- to 34-kDa myristoylated protein which induces downregulation of CD4 surface levels and enhances virus infectivity. In adult macaques, Nef has been implicated in pathogenesis and disease progression. Both HIV-1 SF2 Nef and SIVmac239 Nef have been shown to associate with a cellular serine/threonine kinase. We tested five functional Nef isolates to examine whether this kinase association is a property conserved among different isolates. HIV-1 SF2 and 248 and SIVmac239 Nef proteins were found associated with the kinase. HIV-1 NL4-3 and 233 Nef proteins were found weakly associated or not associated with the kinase. All five Nef isolates efficiently downregulated CD4 cell surface expression, suggesting that the association with this cellular kinase is not required for Nef to downregulate CD4. Comparison of the SF2 and NL4-3 isolates shows a differential ability of Nef to enhance infectivity that suggests a possible correlation between kinase association and enhancement of infectivity.

Characterization of Nef-induced CD4 T cell proliferation.

Studies on Nef, a regulatory protein encoded by human immunodeficiency virus (HIV), suggest it plays an important role in HIV pathogenesis. Previously, we reported that Nef binds to class II MHC antigens and induces proliferation of human peripheral blood mononuclear cells (PBMC). Herein, we further characterize PBMC responses to Nef. Polyclonal antisera generated against Nef synthetic peptides blocked proliferation. Responses were T cell-specific and required antigen-presenting cells (APC). T cells responded in the presence of paraformaldehyde-inactivated APC, suggesting that Nef is presented in an unprocessed form. Nef-stimulated cells produced IL 2 and IFN gamma, products of T helper-1 cells. Thus, Nef has superantigen properties in that it binds to MHC class II antigens, does not need processing to be presented by APC, and activates T cells, causing proliferation and production of the T helper 1 cytokines, IL 2 and IFN gamma. The identification of an HIV protein that activates T cells is of considerable interest, given that HIV replicates in T cell blasts but not in quiescent cells.

Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization.

Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.

Purification of an Escherichia coli-expressed Nef protein from the human immunodeficiency virus-type 2.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.

Cellular localization of Nef expressed in persistently HIV-1-infected low-producer astrocytes.

OBJECTIVES: The characterization and localization of HIV-1 Nef highly expressed in permanently infected astrocytes (TH4-7-5) as a model for latent infection of human brain cells. DESIGN: Immunochemical methods are an appropriate tool to investigate expression and localization of cellular proteins. METHODS: Nef expression was analysed by Western blot and immunoperoxidase staining using a panel of monoclonal and polyclonal antibodies. Cellular localization studies were performed by indirect immunofluorescence and subcellular fractionation of TH4-7-5 cells. Myristoylation of Nef was investigated by immunoprecipitation of [3H]myristic acid-labelled cell extract. TH4-7-5 nef gene was cloned and amplified by polymerase chain reaction and the nef nucleotide sequence analysed. RESULTS: Reactivities of various Nef-specific antibodies with Nef antigen in TH4-7-5 cells were demonstrated by Western blot analysis. Immunofluorescence revealed cytoplasmic perinuclear staining of Nef with most antibodies. However, one monoclonal antibody against amino acids 168-175 of Nef showed intense homogeneous nuclear staining in TH4-7-5 cells. Reactivity of this Nef antibody was blocked with recombinant Nef derived from TH4-7-5 cells. After subcellular fractionation, Nef was detected in nuclear, membrane and cytosolic fractions of TH4-7-5 cells. No myristoylated Nef antigen was detectable, perhaps because of a serine residue at position 2 of the TH4-7-5 nef gene instead of the glycine residue required for myristoylation. CONCLUSIONS: Chronically HIV-1-infected astrocytoma cells with restricted virus production express different antigenic forms of Nef, which can be distinguished by their subcellular localization. Variant subcellular targeting of Nef suggests the existence of multiple activities of Nef within HIV-infected cells.

Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product.

The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.

Expression and cellular localization of the Nef protein from human immunodeficiency virus-1 in stably transfected B-cells.

Nef protein, encoded by the regulatory nef gene of human immunodeficiency virus type 1 (HIV-1), was expressed in the B-cell line Raji. The cells were stably transfected with plasmids containing the nef transcriptional cassette. They expressed Nef with an Mr of 27,000; the yield could be augmented by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The intracellular localization of Nef was analyzed applying immunofluorescence microscopy using a confocal laser scanning microscope. The antigen was stained with a monoclonal antibody directed against the N-terminal part of Nef. The experiments revealed that in non-dividing cells Nef is present both in the cytoplasm and the nucleus while in dividing cells the viral protein is present in the cytoplasm and at the nuclear membrane.

The HIV-1 nef protein does not have guanine nucleotide binding, GTPase, or autophosphorylating activities.

Recombinant HIV-1 Nef proteins with either thr-15 or ala-15 have been constructed and expressed in the T7 bacterial system. From the soluble portion of bacterial lysates both Nef(thr-15) and Nef(ala-15) have been purified to near homogeneity through 6 nondenaturing chromatographic steps in the presence of MgCl2. Neither purified proteins display the previously reported GTP binding activity. Additionally Nef(thr-15) does not have autophosphorylating activity with either [gamma-32P]GTP or [gamma-32P]ATP. Although GTPase activity is present in the preparations of Nef proteins, it does not increase during purification and is attributed to bacterial contaminations.

Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus.

We have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. Spodoptera frugiperda cells infected with the recombinant virus produced a 27-kDa protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the Nef protein by immunoblot analysis. Labeling experiment showed that the recombinant Nef protein was myristoylated. The recombinant Nef protein was purified to near homogeneity by DEAE-Sephacel, phenyl-Sepharose 4B, blue-Sepharose, and Sephadex G-150 column chromatography. No detectable GTP binding activity was observed in the purified recombinant Nef product.

Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1.

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.