Conformational changes in HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion and inactivation

HIV-1 envelope glycoprotein-mediated fusion is driven by the concerted coalescence of the HIV-1 gp41 N- and C-helical regions, which results in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by monitoring the temporal sequence of conformational states of HIV-1 gp41 during the course of HIV-1-mediated cell-cell fusion by quantitative video microscopy using reagents that bind to N- and C-helical regions, respectively. Env-expressing cells were primed by incubation with target cells at different times at 37 degrees C followed by washing. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which we demonstrate here to bind to N-helical gp41 trimers, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed. In contrast, reactivity with 5-helix, which binds to the C-helical region of gp41, increased continuously as a function of time following the priming. The peptide N36(Mut(e,g)) reduced NC-1 monoclonal antibody binding and enhanced 5-helix binding, consistent with the notion that this molecule promotes dissociation of gp41 trimers. This inactivation pathway may be important for the design of entry inhibitors and vaccine candidates.     

Exposure of the membrane-proximal external region of HIV-1 gp41 in the course of HIV-1 envelope glycoprotein-mediated fusion

The membrane-proximal external region (MPER) of HIV-1 gp41 is highly conserved and critical for the fusogenic ability of the virus. However, little is known about the activity of this region in the context of viral fusion. In this study we investigate the temporal exposure of MPER during the course of HIV-1 Env-mediated fusion. We employed the broadly neutralizing monoclonal antibodies 2F5 and 4E10, whose epitopes localize to this region as indicators for accessibility to this region. Time of addition experiments indicated that escape of HIV-1 infection inhibition by 2F5 and 4E10 occurred concomitantly with that of C34, a peptide that blocks the six-helix bundle formation and fusion, which was about 20 min later than escape of inhibition by the mAb b12 that blocks CD4-gp120 attachment. We also probed accessibility of the MPER region on fusion intermediates by measuring the binding of the monoclonal antibodies at different time points during the fusion reaction. Immunofluorescence and in-cell Western assays showed that binding of 2F5 and 4E10 decreased upon triggering HIV-1 Env-expressing cells with appropriate target cells. Addition of C34 did not counteract the loss of antibody binding, suggesting that changes in exposure of MPER occur independently of six-helix bundle formation.     

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).     

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system

We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.     

Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models

Background

Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine.

Methods

We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope.

Results

We applied our method to ID₅₀ neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF)?>?6), a subset of which were experimentally confirmed using site-directed mutagenesis.

Conclusions

Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.

     

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.     

Characterization of monoclonal antibodies to human immunodefiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli

Abstract Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of λ-BH10 cDNA of the human immunowdeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 [1, 2]. Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherochia coli encoding ten distinct segments of the env proteins [3]. In comparison, another mouse MAb, M25 [4], a human MAb directed against gp41, which was produced by the xeno hydridoma line 3D6 [5, 6] and a pool of human patient sera containing antibodies to HIV-1 were tested. We were able to demonstrate that the epitopes recognized by our MAbs are located betweeni arg732 and ser759 [7] of the HIV-1 env glycoprotein gp160 of HTLV-III strain B. M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 [4]. The human MAb against gp41, 3D6 [5, 6] reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids. The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants locatedp between ile474 and leu863. The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes [3]. In this study, we showed that the set of recombinant evr proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins.     

Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.     

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.     

Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41

Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35CCG-N13, N34CCG (a smaller version of N35CCG-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC50 values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6 microg/ml to 60 microg/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.     

Immunological characterization of the sheep prion protein expressed as fusion proteins in Escherichia coli

The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot.     

Kinetic studies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion

Background

Human immunodeficiency virus (HIV) enters target cells by a membrane fusion process that involves a series of sequential interactions between its envelope glycoproteins, the CD4 receptor and CXCR4/CCR5 coreceptors. CD4 molecules are expressed at the cell surface of lymphocytes and monocytes mainly as monomers, but basal levels of CD4 dimers are also present at the cell surface of these cells. Previous evidence indicates that the membrane distal and proximal extracellular domains of CD4, respectively D1 and D4, are involved in receptor dimerization.

Results

Here, we have used A201 cell lines expressing two CD4 mutants, CD4-E91K, E92K (D1 mutant) and CD4-Q344E (D4 mutant), harboring dimerization defects to analyze the role of CD4 dimerization in HIV-1 entry. Using entry assays based on β-lactamase-Vpr or luciferase reporter activities, as well as virus encoding envelope glycoproteins derived from primary or laboratory-adapted strains, we obtained evidence suggesting an association between disruption of CD4 dimerization and increased viral entry efficiency.

Conclusion

Taken together, our results suggest that monomeric forms of CD4 are preferentially used by HIV-1 to gain entry into target cells, thus implying that the dimer/monomer ratio at the cell surface of HIV-1 target cells may modulate the efficiency of HIV-1 entry.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

Temperature-dependent intermediates in HIV-1 envelope glycoprotein-mediated fusion revealed by inhibitors that target N- and C-terminal helical regions of HIV-1 gp41

Peptides derived from the N- (N-HR) and C- (C-HR) terminal heptad repeat regions adjacent to the fusion peptide and transmembrane domains, respectively, of human immunodeficiency virus (HIV)-1 gp41 inhibit HIV-1 viral envelope glycoproteins (Env)-mediated cell fusion specifically. The mechanism of HIV-1 Env-mediated cell fusion and its inhibition by agents that target the N- and C-HR regions was investigated. Priming experiments with Env-expressing cells indicate that the N-HR region but not the C-HR region is exposed by treatment with sCD4 at 31 degrees C, whereas both the N- and C-HR regions are exposed at 37 degrees C.     

Biosynthesis and transport of envelope proteins of Escherichia coli

Bacterial protein synthesis takes place in the cytoplasm, thus periplasmic and outer membrane proteins pass through the cytoplasmic membrane during their dispatch to the cell envelope. The exported proteins are synthesized as precursor that contains an extra amino-terminal sequence of amino-acids. This sequence, termed "signal sequence", is essential for transport of the envelope proteins through the inner membrane and is cleaved during the exportation process. Various hypotheses for the mechanism have been presented, and it is likely that no signal model will be suitable to the export of all cell envelope proteins. This review is focused on the relationship between the cytoplasmic membrane and the precursor form. The physiological state of the membrane - fluidity, membrane potential for instance - is the strategic requirement of exportation process. Precursors can be accumulated in whole cells with various treatments which alter the cytoplasmic membrane. This inhibition of processing is obtained by modification of unsaturated to saturated fatty acids ratio or with phenylethyl alcohol which perturbs the membrane fluidity, with uncoupler agents such as carbonyl cyanide m-chlorophenyl hydrazone which dissipate the proton motive force, or with hybrid proteins which get jamming in the membrane. However, little is known about the early steps of translocation process across the cytoplasmic membrane ; for instance, it is not clear yet whether energy is required for either or both of the first interaction membrane-precursor and the crossing through the membrane. Several studies have recently shown the presence of exportation sites and of proteins which might play a prominent role in the export process, but the mechanism of discrimination between outer membrane proteins and periplasmic proteins is unknown. Considerable work has been done by genetic or biochemical methods and we have now the first lights of the expert mechanism.     

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.