Affinity analysis of differentially expressed genes in hepatocytes expressing HCV core genotype 1b or 3a

Chronic hepatitis C patients display many genotype-specific clinical features of HCV infection. The core proteins encoded by different genotypes dysregulate numerous sets of distinct host genes. In this study we tested the hypothesis that HCV core proteins 1b and 3a would actually act on a limited number of independent cellular players, as well as on several functionally linked gene products. Structural and functional tests identified a core set of host genes dysregulated by HCV core genotypes 1b and 3a. The core proteins of HCV genotypes 1b and 3a target specifically limited sets of functionally related gene products, which may be responsible for the variations in the clinical spectra associated with HCV infection.     

Binding of full-length HIV-1 gp120 to CD4 induces structural reorientation around the gp120 core

Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor, and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.     

Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.     

表达中国流行株HIV-1 gp120外膜蛋白靶细胞模型的建立

利用pDispaly真核表达载体构建了用于表达中国流行株HIV-1 gp120的真核表达质粒pD-120,通过脂质体转染法将其导入人宫颈癌细胞（HeLa）,经G418反复加压筛选,直到细胞不再死亡为止,8代后获得稳定表达中国流行株HIV-1gp120蛋白的靶细胞复制模型HeLa-gp,SDS-PAGE、蛋白印迹与间接免疫荧光分析表明,重组蛋白得到很好表达,并具有良好生物活性。本研究为抗AIDS/HIV治疗用基因工程制剂或靶向药物的活性检测奠定了坚实基础。     

Characterization of the conformational state and flexibility of HIV-1 glycoprotein gp120 core domain

gp120 is key to the human immunodeficiency virus type 1 viral cell entry. Knowledge of the detailed conformational states of gp120 is crucial to intervention, yet the unbound form is still resistant to structural characterization probably because of its flexibility. Toward this goal, we performed molecular dynamics simulations on the wild type gp120 core domain extracted from its ternary crystal structure and on a modeled mutant, S375W, that experimentally has a significantly different phenotype from the wild type. Although the mutant retained a bound-like conformation, the wild type drifted to a different conformational state. The wild type beta strands 2 and 3 of the bridging sheet were very mobile and partially unfolded, and the organization among the inner and outer domains and beta strands 20 and 21 of the bridging sheet, near the mutation site, was more open than in the bound form, although the overall structure was maintained. These differences were apparently a result of the strengthening of the hydrophobic core in the mutant. This stabilization further explains the experimentally significantly different thermodynamic properties between the wild type and the mutant. Taken together, our results suggest that the free form, although different from the bound state, shares many of the bound structural features. The observed loss of freedom near the binding site, rather than the previously hypothesized more dramatic conformational transition from the unbound to the bound state, appears to be the major contributor to the large entropy cost for the CD4 binding to the wild type.     

Elicitation of neutralizing antibodies by intranasal administration of recombinant vesicular stomatitis virus expressing human immunodeficiency virus type 1 gp120

Recombinant viral vectors are useful tools for AIDS vaccine development. However, expression of HIV-1 envelope genes using viral vectors has not been successful in the induction of potent neutralizing antibodies in vivo. We took advantage of the strong immunogenicity of vesicular stomatitis virus (VSV)-based vector and expressed HIV-1 HXB2 gp120 gene in the recombinant VSV. Our results showed that HIV-1 gp120 protein expressed by the recombinant VSV retained the native conformation of the protein to some degree and was recognized by two well-characterized broad anti-HIV-1 neutralizing monoclonal antibodies b12, 2G12. We further showed that only one time intranasal immunization with the recombinant VSV led to production of anti-HIV-1 anti-sera in mice. In addition, we found that the anti-sera had the ability to neutralize not only HXB2 envelope-pseudotyped HIV-1 viruses but also HIV-1 pseudotyped viruses with JRFL envelopes. These results suggest that HIV-1 gp120 expressed by the recombinant VSV, in combination with the route of intranasal administration, is an effective strategy to evaluate the immunogenicity of HIV-1 envelope protein and its variants in mice.     

Recognition of HIV glycoprotein gp120 by T cells. Role of monocyte CD4 in the presentation of gp120

The HIV envelope glycoprotein gp120 binds with high affinity to CD4 and is responsible for the tropism of HIV for CD4+ T cells and monocytes. Efforts to develop HIV vaccines have focused on gp120 and, therefore, a detailed molecular understanding of human immune responses to gp120 is essential. In this report, we have used human T cell clones specific for gp120 to examine the processing and presentation of gp120 to T cells. In particular, we examined the role of the CD4 that is expressed at low levels on the surfaces of human monocytes in the presentation of gp120 by monocytes. The presentation of gp120 to gp120-specific human T cell clones was blocked by pretreatment of monocytes with anti-CD4 mAb. Blocking of monocyte CD4 with anti-CD4 did not inhibit presentation of other Ag or of synthetic peptides representing epitopes within gp120 recognized by gp120-specific T cell clones. These results indicated that the anti-CD4-mediated inhibition occurred at the level of the monocyte, was specific for the gp120 response, and was operative at the initial Ag uptake phase of the Ag-processing pathway. Definitive confirmation that monocyte CD4 functions in the initial uptake step of the gp120-processing pathway was obtained by using soluble CD4 to block the interaction of gp120 with monocyte CD4. These results demonstrate that gp120 expressed by human monocytes plays an important role in the initial uptake of gp120 by monocytes and that gp120 taken up via CD4 is subsequently processed to allow for exposure of epitopes recognized by gp120-specific human T cells. At limiting gp120 concentrations, uptake via CD4 is essential for the presentation of gp120.     

Several antigenic determinants exposed on the gp120 moiety of HIV-1 gp160 are hidden on the mature gp120

Mouse mAb reactive to the HIV-1 envelope glycoprotein precursor gp160 of the HTLVIII(B) isolate were characterized in radioimmunoprecipitation and immunoblot tests with the use of HTLVIII(B) isolate as Ag. The reactivities of these mAb were also measured in a capture enzyme immunoassay and in radioimmunoprecipitation assay by using gp160 and gp120 expressed as vaccinia recombinants. Striking differences in exposure of specific epitopes were noted between the gp120 component of the gp160 precursor and the fully processed gp120 in both tests. These conformational rearrangements affecting the gp120 moiety of the HIV-1 envelope glycoprotein might have important implications on its immunogenicity.     

Mechanisms of the killing of cultured hepatocytes by hydrogen peroxide

Mechanisms of H2O2-induced cell injury were explored in primary cultures of rat hepatocytes. Cells prepared from male rats and cultured for 1 day prior to treatment were killed by H2O2 either added directly to the medium at 0.25-2 mM or generated in situ by glucose oxidase (0.25-2 U/ml) or xanthine oxidase (20-120 mM/ml) and 2 mM xanthine. Catalase protected the cells in each case. Lipid peroxidation as measured by the accumulation of malondialdehyde (MDA) preceded the cell death due to H2O2 added directly to the cultures or generated in the medium. The antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and promethazine prevented the accumulation of MDA in both cases and protected the cells treated with H2O2 directly. DPPD and promethazine did not react directly with H2O2. Other antioxidants including butylated hydroxytoluene, vitamin E, and N-propylgallate had varied protective activity against the addition of H2O2 in proportion to their ability to reduce MDA accumulation. In glucose oxidase-treated cultures, DPPD and promethazine prevented the cell killing during the first hour but failed to protect between 1 and 3 h despite prevention of lipid peroxidation. The cell killing between 1 and 3 h in the presence of DPPD was prevented by catalase indicating its dependence upon continued generation of H2O2. Further addition of H2O2 in the presence of DPPD also increased the number of dead cells without lipid peroxidation. The data are consistent with at least two mechanisms of hepatocyte killing by H2O2. The first pathway is prevented by the antioxidants DPPD and promethazine and is very likely related to the peroxidation of membrane phospholipids. The second is independent of lipid peroxidation yet dependent upon the continued presence of H2O2.     

Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.

Virus-specific CD4(+) T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4(+) as well as CD8(+) T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4(+) T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4(+) T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4(+) T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4(+) T cell responses was selective, since vaccine-elicited Ab and CD8(+) T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4(+) T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.     

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors and corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4–gp120 interaction, CD4 antigen–antibody interaction, and gp120 antigen–antibody interaction were 25.45 ± 20.46, 51.22 ± 34.64, and 89.87 ± 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.     

Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.     

Probing the HIV gp120 envelope glycoprotein conformation by NMR

The HIV envelope glycoprotein gp120 plays a critical role in virus entry, and thus, its structure is of extreme interest for the development of novel therapeutics and vaccines. To date, high resolution structural information about gp120 in complex with gp41 has proven intractable. In this study, we characterize the structural properties of gp120 in the presence and absence of gp41 domains by NMR. Using the peptide probe 12p1 (sequence, RINNIPWSEAMM), which was identified previously as an entry inhibitor that binds to gp120, we identify atoms of 12p1 in close contact with gp120 in the monomeric and trimeric states. Interestingly, the binding mode of 12p1 with gp120 is similar for clades B and C. In addition, we show a subtle difference in the binding mode of 12p1 in the presence of gp41 domains, i.e. the trimeric state, which we interpret as small differences in the gp120 structure in the presence of gp41.     

Durable cytotoxic immune responses against gp120 elicited by recombinant SV40 vectors encoding HIV-1 gp120 +/- IL-15

BACKGROUND: A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency. METHODS: SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120. RESULTS: Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with >/= 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses. CONCLUSIONS: Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV.     

Enhanced dynamics of HIV gp120 glycoprotein by small molecule binding

HIV cell entry and infection are driven by binding events to the CD4 and chemokine receptors with associated conformational change of the viral glycoprotein, gp120. Scyllatoxin miniprotein CD4 mimetics and a small molecule inhibitor of CD4 binding, NBD-556, also effectively induce gp120 conformational change. In this study we examine the fluctuation profile of gp120 in context of CD4, a miniprotein mimetic, and NBD-556 with the aim of understanding the effect of ligand binding on gp120 conformational dynamics. Analysis of molecular dynamics trajectories indicate that NBD-556 binding in the Phe 43 cavity enhances the overall mobility of gp120, especially in the outer domain in comparison to CD4 or miniprotein bound complex. Interactions with the more flexible bridging sheet strengthen upon NBD-556 binding and may contribute to gp120 restructuring. The enhanced mobility of D368, E370, and I371 with NBD-556 bound in the Phe 43 cavity suggests that interactions with α3-helix in the outer domain are not optimal, providing further insights into gp120--small molecule interactions that may impact small molecule designs.     

Targeting of HIV gp120 by oligonucleotide-photosensitizer conjugates. Light-induced damages

The effects of the lysosphingolipid, sphingosylphosphorylcholine (SPC), on the cardiac ryanodine receptor (RyR) were examined. The open probability of cardiac RyR incorporated in lipid bilayers was decreased by cytoplasmic, but not lumenal side application of micromolar concentrations of SPC. Modification of channel function was characterized by the appearance of a long-lived closed state in addition to the brief channel closings observed in the presence and absence of SPC. Open channel kinetics and ion conduction properties, however, were not altered by this compound. These results suggest that SPC, a putative second messenger derived from sphingomyelin, may regulate Ca(2+) release from the sarcoplasmic reticulum by modifying the gating kinetics of the RyR.     

Comparative Immunogenicity of HIV-1 gp160, gp140 and gp120 Expressed by Live Attenuated Newcastle Disease Virus Vector

The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein.     

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.     

HIV-1 gp120 induces expression of IL-6 through a nuclear factor-kappa B-dependent mechanism: suppression by gp120 specific small interfering RNA

In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3±2.1 and 11.6±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-κB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKβ inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-κB specific siRNA. We also showed that gp120 could increase the phosphorylation of IκBα. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-κB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-κB pathway.