Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.     

Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus

Abstract Fibronectin (FN) is present in soluble and matrix forms in various body fluids and tissues, and has been shown to bind to several pathogens, including viruses. The interaction of FN with viral proteins of human immunodeficiency virus (HIV-1) was investigated by immunofluorescence technique using a cell line chronically infected with HIV-1 (H9-V). The results of this study showed that FN binds to HIV-1 infected cells. especially at FN concentration of 5 μg/ml. In addition, FN-pentapeptide has shown the ability to bind to HIV-1 infected cells. On the other hand, preincubation with antibodies against FN abolished the binding of FN to HIV-1 infected cells. Finally, FN has shown to bind to HIV-1 glycoproteins, including gp41 and pg120. In contrast, no binding to HIV-1 core proteins, including p15 and p24, was noted. We suggest that FN, in binding HIV-1 particles, may reduce viremia and thus may be involved in the clearance of viral proteins from the cells.     

The effect of HIV-1 Gag myristoylation on membrane binding

During the viral life cycle, an HIV protein, Gag, assembles at the host membrane, specifically at lipid raft regions, at very high concentrations leading to viral particle budding. Gag is post-translationally modified with an N-terminal myristate group which is thought to target Gag to lipid rafts thus aiding in assembly. Here we have analyzed the membrane binding of myristoylated HIV-1 Gag and a non-myristoylated form of HIV-1 Gag to various membrane models. After assessing the extent of myristoylation by HPLC and radiometric assays, we compared membrane binding using fluorescence methods. We found that myristoylated Gag shows a greater than twofold increase in binding affinity to model rafts. A structural model to explain these results is presented.     

Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1

Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man₁₇-BSA and mannan, by -d-GlcNAc₄₇-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human ₁-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal₁₇-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc₄₇-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures.     

Stimulation of human flap endonuclease 1 by human immunodeficiency virus type 1 integrase: possible role for flap endonuclease 1 in 5'-end processing of human immunodeficiency virus type 1 integration intermediates

Human immunodeficiency virus type 1 (HIV-1) DNA integration intermediates consist of viral and host DNA segments separated by a 5-nucleotide gap adjacent to a 5'-AC unpaired dinucleotide. These short-flap (pre-repair) integration intermediates are structurally similar to DNA loci undergoing long-patch base excision repair in mammalian cells. The cellular proteins flap endonuclease 1 (FEN-1), proliferating cell nuclear antigen, replication factor C, DNA ligase I and DNA polymerase delta are required for the repair of this type of DNA lesion. The role of FEN-1 in the base excision repair pathway is to cleave 5'-unpaired flaps in forked structures so that DNA ligase can seal the single-stranded breaks that remain following gap repair. The rate of excision by FEN-1 of 5'-flaps from short- and long-flap oligonucleotide substrates that mimic pre- and post-repair HIV-1 integration intermediates, respectively, and the effect of HIV-1 integrase on these reactions were examined in the present study. Cleavage of 5'-flaps by FEN-1 in pre-repair HIV-1 integration intermediates was relatively inefficient and was further decreased 3-fold by HIV-1 integrase. The rate of removal of 5'-flaps by FEN-1 from post-repair HIV-1 integration intermediates containing relatively long (7-nucleotide) unpaired 5'-tails and short (1-nucleotide) gaps was increased 3-fold relative to that seen with pre-repair substrates and was further stimulated 5- to 10-fold by HIV-1 integrase. Overall, post-repair structures were cleaved 18 times more effectively in the presence of HIV-1 integrase than pre-repair structures. The site of cleavage was 1 or 2 nucleotides 3' of the branch point and was unaffected by HIV-1 integrase. Integrase alone had no detectable activity in removing 5'-flaps from either pre- or post-repair substrates.     

A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.     

融合蛋白与病毒入膜机制研究进展

包膜病毒感染细胞的第一步即病毒与靶细胞膜的融合,它由病毒包膜上的融合蛋白诱发,融合蛋白与受体分子相互作用后暴露出融合肽,它伸向靶膜使两膜紧密接近后,多肽周围的脂质分子进一步重排,通过中间态最后发生融合,本文将介绍近年来病毒融合蛋白及入膜机制研究进展。     

Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox

Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-beta-cyclodextrin (MbetaCD) or 2-hydroxypropyl-beta-cyclodextrin (HbetaCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MbetaCD or HbetaCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.     

Effects of long-chain acyl carnitine on membrane fluidity of human erythrocytes

Amphiphilic compounds such as long-chain acyl carnitines accumulate in ischemic myocardium and potentially contribute to the myocardial damage. To characterize alterations in membrane molecular dynamics produced by palmitoylcarnitine, human erythrocytes were spin-labeled with 5-doxylstearic acid, and membrane fluidity was quantified by measuring the changes in the order parameter derived from ESR spectra. Palmitoylcarnitine induced triphasic alterations in membrane fluidity of human erythrocytes. The membrane fluidity increased for 5 min, then decreased in a concentration-dependent manner. At higher concentrations (100 and 150 μM) of palmitoylcarnitine, membrane fluidity increased again after 30 and 20 min of the incubation, respectively. Addition of 2.8 mM CaCl₂ resulted in a significant decrease in membrane fluidity and enhanced the alterations in membrane fluidity caused by palmitoylcarnitine. The results suggest that alterations in molecular dynamics are one mechanism through which long-chain acyl carnitine could play an important role in ischemic injury.     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞膜脂流动性的影响

目的探讨分叉双歧杆菌的完整肽聚糖（WPG）对巨噬细胞膜脂流动性的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,再用细胞膜磷脂荧光探针标记细胞,最后采用激光共聚焦显微镜结合激光漂白后荧光恢复技术检测巨噬细胞的膜脂流动性。结果WPG刺激组反映小鼠腹腔巨噬细胞膜脂流动性的平均荧光恢复率明显高于对照组（P〈0.01）。结论分叉双歧杆菌的完整肽聚糖可提高巨噬细胞膜脂流动性。     

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.     

Structural and Functional Properties of the Membranotropic HIV-1 Glycoprotein gp41 Loop Region Are Modulated by Its Intrinsic Hydrophobic Core

The gp41 disulfide loop region switches from a soluble state to a membrane-bound state during the human immunodeficiency virus type 1 (HIV-1) envelope-mediated membrane fusion process. The loop possesses a hydrophobic core at the center of the region with an unusual basic residue (Lys-601). Furthermore, two loop core mutations, K601A and L602A, are found to inhibit HIV-1 infectivity while keeping wild type-like levels of the envelope, implying that they exert an inhibitory effect on gp41 during the membrane fusion event. Here, we investigated the mode of action of these mutations on the loop region. We show that the K601A mutation, but not the L602A mutation, abolished the binding of a loop-specific monoclonal antibody to a loop domain peptide. Additionally, the K601A, but not the L602A, impaired disulfide bond formation in the peptides. This was correlated with changes in the circular dichroism spectrum imposed by the K601A mutation. In the membrane, however, the L602A, but not the K601A, reduced the lipid mixing ability of the loop peptides, which was correlated with decreased α-helical content of the L602A mutant. The results suggest that the Lys-601 residue provides a moderate hydrophobicity level within the gp41 loop core that contributes to the proper structure and function of the loop inside and outside the membrane. Because basic residues are found between the loop Cys residues of several lentiviral fusion proteins, the findings may contribute to understanding the fusion mechanism of other viruses as well.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.     

糖皮质激素对肥大细胞胞膜流动性的影响

目的:研究糖皮质激素(皮质酮)对肥大细胞胞膜流动性的快速作用。方法:采用荧光偏振法检测膜流动性,检测不同浓度皮质酮对肥大细胞膜流动性的快速影响以及加用糖皮质激素受体拮抗剂RU38486看其是否影响皮质酮对肥大细胞膜流动性的快速作用。结果:与阴性对照组比较,皮质酮能够在7min内剂量依赖性地快速降低肥大细胞胞膜流动性,稳定肥大细胞胞膜(P0.01);加用糖皮质激素受体拮抗剂RU38486后能部分阻断皮质酮对肥大细胞膜流动性的快速作用(P0.01)。结论:糖皮质激素能够快速降低肥大细胞胞膜流动性,稳定肥大细胞胞膜,这一作用可能是糖皮质激素快速抑制肥大细胞脱颗粒非基因组机制作用的靶点之一。     

Cepharanthine inhibited HIV-1 cell–cell transmission and cell-free infection via modification of cell membrane fluidity

The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.