CutC is induced late during copper exposure and can modify intracellular copper content in Enterococcus faecalis

The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX_nHX_mHP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.     

用Far-Western印迹技术筛选人肝组织中与乙肝病毒表面抗原PreS1相互作用的蛋白

目的:利用Far-Western印迹技术从正常人肝组织中筛选乙型肝炎病毒(HBV)表面抗原PreS1结合蛋白,为阐明HBV的感染致病机理提供依据。方法:提取正常人肝组织细胞膜蛋白,双向电泳展示后转膜,对表达纯化获得的PreS1的重要片段与GST的融合蛋白PreS/1-48myr-GST进行Far-Western印迹实验,对筛选获得的蛋白点切胶,质谱鉴定。结果:对PreS/1-48myr-GST融合蛋白进行Far-Western-2D筛选,共获得22个蛋白点,经质谱鉴定获得15个候选相互作用蛋白,其中膜蛋白Ezrin可能在HBV感染致病过程中具有重要作用。结论:Ezrin蛋白能够与乙肝病毒表面抗原PreS1结合,其在HBV感染致病过程中的重要作用值得探索。     

Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region.

Hepatitis B virus particles contain three related viral envelope proteins, the small, middle, and large S (surface) proteins. All three proteins contain the small S amino acid sequence at their carboxyl terminus. It is not clear which of these S proteins functions as the viral attachment protein, binding to a target cell receptor and initiating infection. In this report, recombinant hepatitis B surface antigen (rHBsAg) particles, which contain only virus envelope proteins, were radioactively labeled, and their attachment to human liver membranes was examined. Only the rHBsAg particles containing the large S protein were capable of directly attaching to liver plasma membranes. The attachment was saturable and could be prevented by competition with unlabeled particles or by a monoclonal antibody specific for the large S protein. In the presence of polymerized human serum albumin, both large and middle S protein-containing rHBsAg particles were capable of attaching to the liver plasma membranes. Small S protein-containing rHBsAg particles were not able to attach even in the presence of polymerized human serum albumin. These results indicate that the large S protein may be the viral attachment protein for hepatocytes, binding directly to liver plasma membranes by its unique amino-terminal (pre-S1) sequence. These results also indicate that polymerized human serum albumin or a similar molecule could act as an intermediate receptor, attaching to liver plasma membranes and to the amino acid sequence (pre-S2) shared by the middle and large S proteins but not contained in the small S protein.     

乙型肝炎病毒PreS2蛋白研究进展

乙型肝炎病毒(HBV)外膜蛋白包括S蛋白、PreS1蛋白和PreS2蛋白,它们是HBV包膜的主要成分,可诱导机体产生相应的抗体。PreS2蛋白在HBV侵入肝细胞的过程中起着非常重要的作用,对于防治HBV的感染有重要意义。我们简要综述了有关PreS2蛋白的研究,介绍了PreS2蛋白的各项功能。     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

Fusion-defective gibbon ape leukemia virus vectors can be rescued by homologous but not heterologous soluble envelope proteins

Murine leukemia virus (MLV)-derived envelope proteins containing alterations in or adjacent to the highly conserved PHQ motif present at the N terminus of the envelope surface subunit (SU) are incorporated into vector particles but are not infectious due to a postbinding block to viral entry. These mutants can be rendered infectious by the addition of soluble receptor-binding domain (RBD) proteins in the culture medium. The RBD proteins that rescue the infectivity of these defective MLV vectors can be derived from the same MLV or from other MLVs that use distinct receptors to mediate entry. We have now constructed functional immunologically reactive gibbon ape leukemia virus (GALV) envelope proteins, tagged with a feline leukemia virus (FeLV)-derived epitope tag, which are efficiently incorporated into infectious particles. Tagged GALV envelope proteins bind specifically to cells expressing the phosphate transporter protein Pit1, demonstrating for the first time that Pit1 is the binding receptor for GALV and not a coreceptor or another type of GALV entry factor. We have also determined that GALV particles bearing SU proteins with an insertion C-terminal to the PHQ motif (GALV I(10)) bind Pit1 but fail to infect cells. Incubation with soluble GALV RBD renders GALV I(10) particles infectious, whereas incubation with soluble RBDs from MLV or FeLV-B does not. This finding is consistent with the results obtained by Lauring et al. using FeLV-T, a virus that employs Pit1 as a receptor but requires soluble FeLV RBD for entry. MLV and GALV RBDs are not able to render FeLV-T infectious (A. S. Lauring, M. M. Anderson, and J. Overbaugh, J. Virol. 75:8888-8898, 2001). Together, these results suggest that fusion-defective FeLV-T and GALV are restricted to homologous RBD rescue of infectivity.     

Phage display selection on whole cells yields a small peptide specific for HCV receptor human CD81

The human CD81 (hCD81), the most recently proposed receptor of hepatitis C virus (HCV), can especifically bind to HCV envelope glycoprotein2 (E2). In this study, hCD81-expressing murine NIH/3T3 cells were used to select hCD81-binding peptides from a phage displayed nonapeptide library (PVⅢ9aaCys). Eighteen of the 75 clones selected from the library showed specific binding to the hCD81-expressing NIH/3T3 cells by enzyme linked immunosorbent assay (ELISA) and competitive inhibition test. Twelve out of the 18 clones shared the amino acid motif SPQYWTGPA. Sequence comparison of the motif showed no amino acid homology with the native HCV E2. The motif-containing phages could competitively inhibit the ability of HCV E2 binding to native hCD81-expressing MOLT-4 cells, and induce HCV E2 specific immune response in vivo. These results suggest that the selected motif SPQYWTGPA should be a mimotope of HCV E2 to bind to hCD81 molecules. Our findings cast new light on developing HCV receptor antagonists.     

Assessing the role of the glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) three-finger domain in binding lipoprotein lipase

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.     

Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.     

Initial Binding of Murine Leukemia Virus Particles to Cells Does Not Require Specific Env-Receptor Interaction

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.     

N-terminal myristylation of HBV preS1 domain enhances receptor recognition.

The N-terminal portion of the large envelope protein of the human hepatitis B virus (HBV), the preS1 domain, plays a fundamental role in cell attachment and infectivity. Recent investigations have suggested that myristylation of preS1 Gly2 residue is essential for viral infectivity, but the importance of this post-translational modification on HBV-receptor interaction has not been elucidated completely. In this study we produced, using stepwise solid-phase chemical synthesis, the entire preS1[1-119] domain (adw2 subtype), and compared its receptor binding activity with the myristylated form, myristyl-preS1[2-119] in order to define the importance of fatty acid modification. Both synthetic proteins were fully characterized in terms of structural identity using TOF-MALDI mass spectrometry and analysis of tryptic fragments. Circular dichroism measurements indicated a low content of ordered structure in the preS1 protein, while the propensity of the myristylated derivative to assume a conformationally defined structure was more evident. HBV-receptor binding assays performed with plasma membranes preparations from the hepatocyte carcinoma cell line HepG2 clearly showed that the preS1[1-119] domain recognizes the HBV receptor, and confirmed that binding is occurring through the 21-47 region. The myristylated derivative recognized HBV receptor preparations with higher affinity than the preS1 domain, suggesting that the conformational transitions induced in the preS1 moiety by fatty acid post-translational modification are important for efficient attachment of viral particles to HBV receptors.     

Rabies virus binding to an acetylcholine receptor alpha-subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.     

Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus

Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences.     

The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity

The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.     

An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione S-transferase (GST), expressed in Escherichia coli, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST-pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.     

The highly conserved glycan at asparagine 260 of HIV-1 gp120 is indispensable for viral entry

Carbohydrate-binding agents bind to the N-glycans of HIV-1 envelope gp120 and prevent viral entry. Carbohydrate-binding agents can select for mutant viruses with deleted envelope glycans. Not all glycosylation motifs are mutated to the same extent. Site-directed mutagenesis revealed that deletions destroying the highly conserved (260)NGS(262) glycosylation motif resulted in non-infectious virus particles. We observed a significant lower CD4 binding in the case of the N260Q mutant gp120 virus strains, caused by a strikingly lower expression of gp120 and gp41 in the virus particle. In addition, the mutant N260Q HIV-1 envelope expressed in 293T cells was unable to form syncytia in co-cultures with U87.CD4.CXCR4.CCR5 cells, due to the lower expression of envelope protein on the surface of the transfected 293T cells. The detrimental consequence of this N-glycan deletion on virus infectivity could not be compensated for by the creation of novel glycosylation sites near this amino acid, leaving this uncovered envelope epitope susceptible to neutralizing antibody binding. Thus, the Asn-260 glycan in the gp120 envelope of HIV-1 represents a hot spot for targeting suicidal drugs or antibodies in a therapeutic effort to efficiently neutralize a broad array of virus strains.     

Unmasking ligand binding motifs: identification of a chemokine receptor motif by NMR studies of antagonist peptides

Determining the critical structural features a ligand must possess in order to bind to its receptor is of key importance to the understanding of vital biological processes and to the rational design of small molecule therapeutics to modulate receptor function. We have developed a general strategy for determining such ligand binding motifs using low temperature NMR structures of peptides with the desired receptor binding properties. This approach has been successfully applied to determine a binding motif for the chemokine receptor CXCR4. The motif identified provides a detailed guide for the design of small molecule antagonists against CXCR4, which are much sought after to aid in the treatment of a number of conditions including human immunodeficiency virus type 1 infection and a variety of cancers.     

The use of phage display technique for the isolation of androgen receptor interacting peptides with (F/W)XXL(F/W) and FXXLY new signature motifs

Early studies suggested that the signature motif, LXXLL, within steroid hormone receptor p160 coregulators may play important roles for the mediation of receptor-coregulator interaction. Interestingly, several androgen receptor (AR) coregulators, such as ARA70 and ARA55, may not use such a unique motif to mediate their coregulator activity. Here we apply the phage display technique to identify some new signature motifs, (F/W)XXL(F/W) and FXXLY (where F is phenylalanine, W is tryptophan, L is leucine, Y is tyrosine, and X is any amino acid) that can influence the interaction between AR and AR coregulators. Sequence analyses found that several AR coregulators, such as ARA70, ARA55, ARA54, and FHL2, contain FXXL(F/Y) motifs. Both glutathione S-transferase pull-down assays and transient transfection reporter assays demonstrate that these AR coregulators may use the FXXL(F/Y) motif to interact with AR and exert their AR coregulator activity. Exchanging the amino acid of Phe, Trp, or Tyr in this newly identified signature motif cluster may influence these peptides to interact with AR. The motif-containing peptides, as well as ARA70 or ARA54, may require selective flanking sequences for the better interaction with AR. In addition to influencing the AR transactivation, these motifs in AR-interacting peptides/proteins were also able to influence the AR N-/C-terminal interaction. Together, our data suggest that AR interacting peptides and/or AR coregulators may utilize the (F/W)XXL(F/W) and FXXLY motifs to mediate their interaction with AR and exert their influences on the AR transactivation.     

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.