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1.
Vaccine ; 24(25): 5269-76, 2006 Jun 19.
Article in English | MEDLINE | ID: mdl-16713032

ABSTRACT

The HIV-1 matrix protein p17 is a structural protein essential in the life cycle of HIV, by acting as a virokine/immunomodulator that supports viral replication and spreading. The presence of p17-specific antibodies and CTL responses correlates with slower progression to AIDS. Intranasal vaccination with p17 and the TLR2/6 agonist MALP-2 stimulates strong humoral and cellular immune responses at systemic and mucosal levels. The antibodies blocked p17 binding to its receptor, which is a critical step for the exertion of its virokine activity. Our results suggest that p17 and MALP-2 are attractive candidates for incorporation in mucosal vaccines against HIV/AIDS.


Subject(s)
AIDS Vaccines/administration & dosage , Adjuvants, Immunologic/administration & dosage , Gene Products, gag/administration & dosage , HIV Antigens/administration & dosage , HIV-1/immunology , Oligopeptides/administration & dosage , Viral Proteins/administration & dosage , AIDS Vaccines/immunology , Administration, Intranasal , Animals , Female , Gene Products, gag/chemical synthesis , Gene Products, gag/chemistry , Gene Products, gag/immunology , HIV Antibodies/blood , HIV Antibodies/metabolism , HIV Antigens/chemistry , HIV Antigens/immunology , HIV Infections/immunology , HIV Infections/prevention & control , Humans , Immunity, Mucosal , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/blood , Lipopeptides , Mice , Mice, Inbred BALB C , Oligopeptides/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Toll-Like Receptor 2 , Viral Proteins/chemical synthesis , Viral Proteins/chemistry , Viral Proteins/immunology , gag Gene Products, Human Immunodeficiency Virus
2.
Proc Natl Acad Sci U S A ; 101(32): 11587-92, 2004 Aug 10.
Article in English | MEDLINE | ID: mdl-15280532

ABSTRACT

The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this "myristoyl switch" hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.


Subject(s)
Gene Products, gag/chemical synthesis , Myristates/chemical synthesis , Viral Proteins/chemical synthesis , Cell Membrane/metabolism , Dimerization , Gene Products, gag/chemistry , Gene Products, gag/metabolism , HIV Antigens/chemistry , HIV Antigens/metabolism , Protein Binding , Protein Conformation , Protein Denaturation , Spectrum Analysis , Viral Proteins/chemistry , Viral Proteins/metabolism , gag Gene Products, Human Immunodeficiency Virus
3.
J Virol ; 74(22): 10822-6, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11044131

ABSTRACT

Based on the human immunodeficiency virus type 1 (HIV-1) gag gene, subgenomic reporter constructs have been established allowing the contributions of different cis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimized gag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.


Subject(s)
Gene Expression , Gene Products, rev/metabolism , HIV-1/genetics , HIV-1/metabolism , Protein Sorting Signals/genetics , Base Sequence , Cell Nucleus/metabolism , Gene Products, gag/chemical synthesis , Gene Products, gag/genetics , Gene Products, gag/metabolism , Gene Products, rev/genetics , Humans , Molecular Sequence Data , RNA Splicing , RNA, Messenger/metabolism , Tumor Cells, Cultured , rev Gene Products, Human Immunodeficiency Virus
4.
AIDS Res Hum Retroviruses ; 16(13): 1259-67, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10957723

ABSTRACT

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Capsid Proteins , Capsid/immunology , Gene Products, gag/immunology , Viral Proteins , Amino Acid Sequence , Animals , Capsid/chemical synthesis , Capsid/chemistry , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Female , Gene Products, gag/chemical synthesis , Gene Products, gag/chemistry , Gene Products, vpr/immunology , Humans , Kinetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/immunology , Precipitin Tests , Protein Precursors/immunology , Surface Plasmon Resonance/methods , gag Gene Products, Human Immunodeficiency Virus
5.
Hybridoma ; 18(4): 305-14, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10571260

ABSTRACT

The object of this study was to examine the possibility of immunotherapy using anti-human immunodeficiency virus type 1 (HIV-1) p17-derivative peptide monoclonal antibody (MAb), namely MAb 8H10. Previously we established MAb 8H10, and further characterization revealed that it inhibited the viral multiplication of the HIV-1-infected MT-4 cells, and that the inhibitory mechanism related to the decrease of p17 DNA of the infected cells. In the present study, based on the assumption that Hybridoma 8H10 (Hyb 8H10) is a source of this MAb, we examined how Hyb 8H10 influences the infected cells when the two are co-cultured using the transwell or by mixed culture. Hyb 8H10 did not influence the cell growth or viability of MT-4 cells, and MAb 8H10 was transferred to the cluster dish containing the infected cells. Furthermore, Hyb 8H10 could produce MAb 8H10 even when co-cultured with the infected MT-4 cells. However, the infectivity of the supernatant of the infected cells was temporarily enhanced when Hyb 8H10 was co-cultured without MAb 8H10, though it gradually reduced according to the increase in MAb 8H10 produced by Hyb 8H10. Though there were some problems, MAb 8H10 proved to be a strong candidate for immunotherapy against HIV.


Subject(s)
Antibodies, Monoclonal/pharmacology , Gene Products, gag/immunology , HIV Antibodies/immunology , HIV Antibodies/pharmacology , HIV Antigens/immunology , HIV-1/physiology , Viral Proteins , Animals , Antibody Specificity , Cell Division/drug effects , Coculture Techniques , DNA, Viral/analysis , DNA, Viral/biosynthesis , DNA, Viral/drug effects , Dose-Response Relationship, Drug , Gene Products, gag/chemical synthesis , Gene Products, gag/genetics , HIV Antigens/genetics , HIV-1/genetics , HIV-1/immunology , Humans , Hybridomas/immunology , Hybridomas/virology , Mice , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Time Factors , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/virology , Virus Replication/drug effects , gag Gene Products, Human Immunodeficiency Virus
6.
J Virol ; 73(5): 4257-65, 1999 May.
Article in English | MEDLINE | ID: mdl-10196322

ABSTRACT

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.


Subject(s)
Capsid/immunology , Epitopes, T-Lymphocyte/immunology , Gene Products, gag/immunology , Infectious Anemia Virus, Equine/immunology , T-Lymphocytes, Helper-Inducer/immunology , Viral Core Proteins/immunology , Viral Matrix Proteins/immunology , Amino Acid Sequence , Animals , Capsid/chemical synthesis , Carrier State , Cell Division , Gene Products, gag/chemical synthesis , Horses , Leukocytes, Mononuclear/cytology , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/immunology , Recombinant Fusion Proteins/immunology , Viral Core Proteins/chemical synthesis , Viral Matrix Proteins/chemical synthesis
7.
Anal Biochem ; 260(2): 212-7, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9657880

ABSTRACT

Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X2- Cys-X4-His-X4-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc. Scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc. At pH 6.0, one site is shown to bind zinc strongly with a binding constant Kb = 3.25 x 10(5) M-1 and the second site has a Kb = 1.8 x 10(5) M-1. The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold. The Hill coefficient for this negative cooperativity is 0.9. A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc. This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants.


Subject(s)
Capsid Proteins , Capsid/chemistry , Gene Products, gag/chemistry , HIV-1/chemistry , Nucleocapsid/chemistry , Viral Proteins , Amino Acid Sequence , Capsid/metabolism , Electrophoresis, Capillary/methods , Gene Products, gag/chemical synthesis , Gene Products, gag/metabolism , HIV-1/metabolism , Kinetics , Molecular Sequence Data , Nucleocapsid/chemical synthesis , Nucleocapsid/metabolism , Point Mutation , Zinc/metabolism , Zinc Fingers , gag Gene Products, Human Immunodeficiency Virus
8.
J Pept Res ; 50(4): 269-78, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9352465

ABSTRACT

Specific interactions between the 72-amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non-natural amino acid, (S)-beta-(9-acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three-dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV-1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein-nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.


Subject(s)
Acridines/chemistry , Alanine/analogs & derivatives , Capsid Proteins , Capsid/chemistry , Capsid/metabolism , Fluorescent Dyes/chemical synthesis , Gene Products, gag/chemistry , Gene Products, gag/metabolism , HIV-1/chemistry , RNA, Viral/metabolism , Viral Proteins , Alanine/chemistry , Amino Acid Sequence , Binding, Competitive , Capsid/chemical synthesis , Dimerization , Electrophoresis, Agar Gel , Gene Products, gag/chemical synthesis , HIV-1/metabolism , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/metabolism , Peptides/chemical synthesis , Peptides/metabolism , Protein Binding , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , gag Gene Products, Human Immunodeficiency Virus
9.
Virology ; 226(1): 102-12, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8941327

ABSTRACT

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.


Subject(s)
Antigens, Viral/immunology , Gene Products, env/immunology , Gene Products, gag/immunology , Gene Products, pol/immunology , Gene Products, tax/immunology , HLA-B Antigens/immunology , Human T-lymphotropic virus 1/immunology , Retroviridae Proteins, Oncogenic/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Female , Gene Products, env/chemical synthesis , Gene Products, gag/chemical synthesis , Gene Products, pol/chemical synthesis , Gene Products, tax/chemical synthesis , HLA-B Antigens/genetics , Humans , Mice , Mice, Inbred C3H , Mice, Inbred ICR , Mice, Transgenic , Peptides/chemical synthesis , Peptides/immunology , Retroviridae Proteins, Oncogenic/chemical synthesis , gag Gene Products, Human Immunodeficiency Virus
10.
Biochem Biophys Res Commun ; 224(1): 191-8, 1996 Jul 05.
Article in English | MEDLINE | ID: mdl-8694811

ABSTRACT

The zinc-bound form of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, p7, aggregates into particles visible by electron microscopy. The HIV primer tRNA(Lys,3) forms similar high molecular weight complexes with p7 that are also detected by gel mobility shift assays. RNA oligonucleotides of the three stem-loop structures in tRNA(Lys,3) were assayed for the competitive inhibition of p7-tRNA(Lys,3) binding by the intensities of free tRNA(Lys,3) bands on native gels. This reveals that the p7 binds specifically to the central domain of tRNA(Lys,3) where the D and T psi C loops come together, but not the anticodon stem-loop.


Subject(s)
Capsid Proteins , Capsid/chemical synthesis , Capsid/metabolism , DNA Primers/metabolism , Gene Products, gag/chemical synthesis , Gene Products, gag/metabolism , HIV/physiology , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer, Lys/biosynthesis , Viral Proteins , Base Sequence , Binding Sites , Capsid/ultrastructure , DNA Primers/chemistry , Gene Products, gag/ultrastructure , Humans , Microscopy, Electron , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , RNA, Transfer, Amino Acyl/biosynthesis , RNA, Transfer, Amino Acyl/ultrastructure , RNA, Transfer, Lys/ultrastructure , Restriction Mapping , gag Gene Products, Human Immunodeficiency Virus
11.
Biochemistry ; 35(16): 5175-82, 1996 Apr 23.
Article in English | MEDLINE | ID: mdl-8611501

ABSTRACT

The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.


Subject(s)
Capsid Proteins , Capsid/metabolism , Gene Products, gag/metabolism , HIV-1/metabolism , Viral Proteins , Zinc Fingers , Zinc/metabolism , Amino Acid Sequence , Capsid/chemical synthesis , Computer Simulation , Gene Products, gag/chemical synthesis , Models, Chemical , Molecular Sequence Data , Sequence Deletion , Spectrometry, Fluorescence , Thermodynamics , gag Gene Products, Human Immunodeficiency Virus
12.
FEBS Lett ; 361(1): 85-8, 1995 Mar 13.
Article in English | MEDLINE | ID: mdl-7890046

ABSTRACT

It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.


Subject(s)
Bacteriophages/genetics , Capsid Proteins , Capsid/metabolism , Gene Products, gag/metabolism , HIV-1 , Peptides/metabolism , Recombinant Fusion Proteins/metabolism , Viral Proteins , Amino Acid Sequence , Bacteriophages/metabolism , Capsid/chemical synthesis , Consensus Sequence/genetics , Gene Products, gag/chemical synthesis , Molecular Sequence Data , Peptides/chemical synthesis , Sequence Deletion , Sequence Homology, Amino Acid , Zinc Fingers/genetics , gag Gene Products, Human Immunodeficiency Virus
13.
J Biomol NMR ; 4(2): 153-70, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8019131

ABSTRACT

The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH > or = 5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX2CysX4HEsX4Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz 1H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747 +/- 0.209 A for backbone atoms and 1.709 +/- 0.187 A when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr28, Trp35, Lys37, Lys41 and Lys42 residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.


Subject(s)
Gene Products, gag/chemistry , Moloney murine leukemia virus/chemistry , Protein Conformation , Viral Core Proteins/chemistry , Amino Acid Sequence , Conserved Sequence , Gene Products, gag/chemical synthesis , Hydrogen , Magnetic Resonance Spectroscopy/methods , Mathematics , Models, Molecular , Molecular Sequence Data , Viral Core Proteins/chemical synthesis , Zinc Fingers
14.
C R Acad Sci III ; 313(4): 183-6, 1991.
Article in French | MEDLINE | ID: mdl-1913257

ABSTRACT

We report the synthesis in solid phase of an 18-amino acid peptide that contains the cysteine-rich region of the structural Gag protein of HIV-2. The characterization of this fragment and of its interaction with Zn2+ has been made by one- (1 D) and two-dimensional (2D) NMR techniques and by circular dichroism. Our results suggest that in aqueous solution the complexation produces a significant perturbation in the conformation of this peptide.


Subject(s)
Gene Products, gag/chemical synthesis , HIV-2/isolation & purification , Zinc Fingers , Circular Dichroism , Magnetic Resonance Spectroscopy
15.
Int J Pept Protein Res ; 36(6): 551-8, 1990 Dec.
Article in English | MEDLINE | ID: mdl-1708745

ABSTRACT

The core of retroviruses contains a highly conserved, low molecular weight, basic protein that binds nucleic acids and is essential for genomic RNA packaging. The 56 amino acid protein, NCp10, of Moloney Murine Leukaemia virus (MoMuLV) has the CysX2 CysX4 HisX4 Cys zinc finger-like motif shared by all retrovirus nucleocapsid proteins. The native protein and five modified peptides containing the zinc binding domain were synthesized by solid phase in order to investigate the structural and biochemical role of Zn2+ chelation in MoMuLV NCp10 activity. The purity of the synthetic molecules was verified by HPLC and their sequences were confirmed by amino acid analysis and sequencing in the case of NCp10. Thiol dosage agreed with the theoretical value of free cysteine for all these molecules. Fluorescence measurements performed on synthetic NCp10 and zinc finger fragments showed that the tryptophan quantum yield was Zn2(+)-dependent, allowing a 1:1 stoichiometry for the complex to be determined. The apparent affinity constant of NCp10 for the metal was estimated to be superior to 10(6) M-1. The synthetic protein, in the presence of Zn2+ ions, possesses all the biological properties of NCp10 isolated from virions. It catalyzes both the MoMuLV RNA dimerization and the annealing of the replication primer tRNA(Pro) onto MoMuLV RNA.


Subject(s)
Capsid/chemical synthesis , Gene Products, gag/chemical synthesis , Moloney murine leukemia virus/metabolism , Sulfhydryl Compounds/metabolism , Viral Core Proteins/chemical synthesis , Zinc Fingers , Amino Acid Sequence , Animals , Capsid/genetics , Capsid/metabolism , Fluorescence , Gene Products, gag/metabolism , Mice , Molecular Sequence Data , Moloney murine leukemia virus/genetics , Peptide Fragments/metabolism , RNA/genetics , RNA, Transfer, Pro/genetics , Transcription, Genetic , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Zinc/metabolism
16.
Folia Biol (Praha) ; 36(1): 71-7, 1990.
Article in English | MEDLINE | ID: mdl-2111780

ABSTRACT

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.


Subject(s)
Gene Products, gag/pharmacology , Immunity/drug effects , Lymphocytes/immunology , Peptide Fragments/pharmacology , Viral Core Proteins/pharmacology , Animals , Antibody Formation/drug effects , Cell Line , Erythrocytes/immunology , Gene Products, gag/chemical synthesis , HIV Core Protein p24 , Humans , In Vitro Techniques , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Peptide Fragments/chemical synthesis , Phytohemagglutinins/immunology , Sheep , T-Lymphocytes/immunology , Viral Core Proteins/chemical synthesis
17.
Pept Res ; 1(2): 74-80, 1988.
Article in English | MEDLINE | ID: mdl-2856555

ABSTRACT

The p10 murine leukemia virus (MuLV) protein is a basic single-stranded nucleic acid binding protein encoded by the extreme 3' region of the gag gene of MuLV type C. It contains the Cys-X2-Cys-X4-His-X4-Cys sequence shared by all retroviral gag polyproteins. A similar sequence is found in the gene 32 single-stranded DNA binding protein of bacteriophage T4 and is believed to be the zinc binding region of the protein. Solid phase synthesis of p10 was carried out based on the known primary structure of the native protein, with the exception that the acetamidomethyl (Acm) derivative of cysteine was incorporated at all three cysteine positions. The structure of the synthetic p10 was confirmed by direct amino-acid sequencing, as well as by amino acid analysis and FAB mass spectrometry of endoproteinase Lys-C peptides derived from p10. A Chou and Fasman analysis of the primary sequence predicts that p10 contains 9% beta strand and/or sheet and 36% alpha helix. Circular dichroism experiments carried out on the Acm derivatized peptide gave somewhat different results, in that they suggest that p10 contains approximately 70% random coil, less than 30% beta strand and/or sheet and less than 10% alpha helix. With a Ka of greater than 10(8) M-1 for single-stranded RNA, the synthetic peptide binds as tightly as the p10 protein does when isolated directly from infected HTG-2 cells. The Acm groups can be removed from the synthetic p10 peptide by the use of mercuric acetate, followed by treatment with dithiothreitol to sequester the mercuric ion.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
DNA-Binding Proteins/chemical synthesis , Gene Products, gag/chemical synthesis , Genes, gag/genetics , Leukemia Virus, Murine/chemistry , Amino Acids/analysis , Circular Dichroism , Cysteine/analogs & derivatives , DNA, Single-Stranded , Mass Spectrometry , Poly A/metabolism
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