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1.
J Gen Virol ; 91(Pt 9): 2341-50, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20444994

ABSTRACT

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The interaction was initially established with native proteins by co-immunoprecipitating PUUV nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to bind to the predicted CT of Gc. The importance of Gn- and Gc-CTs to the recognition of RNP was further verified in pull-down assays using soluble peptides with binding capacity to both recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species, suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our previous results, we suggest that the complete hetero-oligomeric (Gn-Gc)(4) spike complex of hantaviruses mediates the packaging of RNP into virions.


Subject(s)
Nucleocapsid Proteins/chemistry , Orthohantavirus/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Antibodies, Neutralizing , Binding Sites/genetics , Chlorocebus aethiops , Orthohantavirus/genetics , Orthohantavirus/immunology , Models, Molecular , Molecular Sequence Data , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/immunology , Peptide Mapping , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Puumala virus/chemistry , Puumala virus/genetics , Puumala virus/immunology , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Ribonucleoproteins/immunology , Sequence Homology, Amino Acid , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Proteins/genetics , Viral Proteins/immunology
2.
APMIS ; 113(7-8): 497-505, 2005.
Article in English | MEDLINE | ID: mdl-16086819

ABSTRACT

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Proteins/chemistry , Proteins/pharmacology , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/toxicity , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , Dimerization , Drug Resistance, Multiple, Bacterial , Enterococcus faecium/drug effects , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Proteins/toxicity , Rana temporaria , Staphylococcus aureus/drug effects , Stereoisomerism
3.
Biol Reprod ; 71(3): 732-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15128595

ABSTRACT

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.


Subject(s)
Antibodies, Monoclonal/pharmacology , Intercellular Signaling Peptides and Proteins/immunology , Intercellular Signaling Peptides and Proteins/metabolism , Mitogens/metabolism , Oocytes/cytology , Amino Acid Sequence , Animals , Bone Morphogenetic Protein 15 , Female , Growth Differentiation Factor 9 , Intercellular Signaling Peptides and Proteins/chemistry , Mice , Mitogens/chemistry , Mitogens/immunology , Molecular Sequence Data , Oocytes/metabolism , Protein Structure, Tertiary , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/metabolism
4.
Viral Immunol ; 15(1): 177-92, 2002.
Article in English | MEDLINE | ID: mdl-11952140

ABSTRACT

Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.


Subject(s)
Epitope Mapping/methods , Epitopes, B-Lymphocyte , Nucleocapsid/immunology , Puumala virus/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Mice , Molecular Sequence Data , Nucleocapsid/chemistry
5.
J Med Virol ; 66(3): 340-50, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11793386

ABSTRACT

Insulin-dependent (type 1) diabetes is characterized by progressive destruction of insulin-producing beta cells probably by autoreactive T lymphocytes. Viral infections, especially those caused by coxsackieviruses, are postulated to play a role in the pathogenesis of the disease in humans. One mechanism by which viral infections could initiate or accelerate diabetogenic processes is "molecular mimicry," induction of antiviral immune responses cross-reacting with epitopes in the beta-cell autoantigens. Tyrosine phosphatases (IA-2, IAR) represent a major target autoantigen in type 1 diabetes. Both humoral and cellular immune responses are directed to the carboxy-terminal (C-terminal) part of the protein. This region has a 5-amino acid sequence identity, followed by five amino acid similarity with the conservative motif in the VP1-protein of enteroviruses (PALTAVETGA/HT), which is a highly immunogenic B- and T-cell epitope in enterovirus infection-induced immune responses. This observation prompted us to investigate potential humoral cross-reactions between immune responses induced by tyrosine phosphatases and enteroviruses. The reactivities of various peptide- and virus-induced rabbit antisera clearly demonstrated that cross-reactions do exist, and in both directions. Using epitope mapping, we were able to show that several diabetes-linked epitopes in IA-2 were also recognized by CBV-4-induced antisera. Immunization of female NOD-mice with formalin-inactivated purified strain of coxsackievirus B4 (CBV-4-E2) induced an immune response that recognized the IA-2/IAR diabetogenic peptide. The results obtained with human paired sera, collected during enterovirus infection, indicated that enterovirus infection in humans may also occasionally induce a humoral response that cross-reacts with IA-2/IAR.


Subject(s)
Autoantigens/immunology , Enterovirus Infections/immunology , Membrane Glycoproteins/immunology , Membrane Proteins/immunology , Protein Tyrosine Phosphatases/immunology , Amino Acid Sequence , Animals , Capsid/immunology , Capsid Proteins , Cross Reactions , Enterovirus A, Human/immunology , Enterovirus B, Human/immunology , Enterovirus Infections/blood , Epitope Mapping , Epitopes, B-Lymphocyte/immunology , Female , Immunization , Islets of Langerhans/immunology , Male , Mice , Mice, Inbred NOD , Molecular Sequence Data , Poliovirus/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Rabbits , Receptor-Like Protein Tyrosine Phosphatases, Class 8
6.
Bioorg Med Chem ; 9(6): 1601-7, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11408179

ABSTRACT

Dimeric derivative of antimicrobial peptide amide Temporin A (TA) was synthesized by using a new branching unit 3-N,N-di(3-aminopropyl)amino propanoic acid (DAPPA), which allows building of the parallelly symmetric alpha-helical structures. Antimicrobial effect of the original peptide amide, its monomeric carboxy (TAc) and novel dimeric (TAd) analogues were tested against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). Both TA and TAd completely inhibited the growth of S. aureus at the concentrations of 5 and 10 microM, respectively, whereas TAc did not show any inhibitory activity. The activities of TAc, TA and TAd correlate directly with the net charges of the molecules, +1, +2 and +4, respectively. Interestingly, TAd displayed antibacterial effect against E. coli at a concentration of 10 microM, where as monomeric TA did not show any activity at concentration as high as 20 microM. The results indicate that the novel structural modification improves the antibacterial properties of Temporin A especially towards Gram-negative bacteria.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Proteins/chemistry , Anti-Bacterial Agents/chemical synthesis , Antimicrobial Cationic Peptides , Escherichia coli/drug effects , Microbial Sensitivity Tests , Proteins/pharmacology , Staphylococcus aureus/drug effects
7.
Acta Biochim Pol ; 48(4): 1185-9, 2001.
Article in English | MEDLINE | ID: mdl-11995990

ABSTRACT

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.


Subject(s)
Anti-Infective Agents/pharmacology , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Proteins/pharmacology , Amino Acid Sequence , Amino Acids/chemistry , Anti-Infective Agents/chemistry , Antifungal Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Blood Coagulation/drug effects , Erythrocytes/metabolism , Hemolysis , Humans , Molecular Sequence Data , Peptide Biosynthesis , Peptides/chemistry , Protein Structure, Tertiary , Proteins/chemistry , Prothrombin/chemistry , Prothrombin/pharmacology , Thromboplastin/chemistry , Thromboplastin/pharmacology
8.
Eur J Biochem ; 267(20): 6212-20, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11012675

ABSTRACT

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.


Subject(s)
Oligopeptides/chemistry , Prostate-Specific Antigen/chemistry , Prostate-Specific Antigen/metabolism , Amino Acid Sequence , Binding Sites , Humans , Kinetics , Oligopeptides/pharmacology , Peptide Library , Serine Endopeptidases/metabolism
9.
FEBS Lett ; 479(1-2): 6-9, 2000 Aug 11.
Article in English | MEDLINE | ID: mdl-10940378

ABSTRACT

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.


Subject(s)
Anti-Bacterial Agents/pharmacology , Peptides/pharmacology , Proteins/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Antimicrobial Cationic Peptides , Circular Dichroism , Drug Resistance, Microbial , Enterococcus faecalis/drug effects , Enterococcus faecium/drug effects , Magnetic Resonance Spectroscopy , Methicillin Resistance , Microbial Sensitivity Tests , Models, Molecular , Peptides/chemistry , Peptides/isolation & purification , Protein Conformation , Proteins/chemistry , Proteins/isolation & purification , Rana temporaria , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Vancomycin Resistance
10.
J Gen Virol ; 81(Pt 7): 1709-18, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10859376

ABSTRACT

Human parechoviruses 1 and 2 (HPEV1 and HPEV2, respectively), formerly known as echoviruses 22 and 23, have been assigned to a novel picornavirus genus on the basis of their distinct molecular and biological properties. To study the immunological characteristics of HPEV1 capsid proteins, antigenic analysis was carried out by a peptide scanning technique, which can be used to identify the immunogenic peptide sequences of a protein. Partially overlapping peptides, representing the capsid of HPEV1, were synthesized using a 12 aa window in a three residue shift and reactivity of rabbit and murine HPEV1 antisera against these peptides were tested. Using this method, an antigenic site in the VP0 polypeptide, recognized by both rabbit and murine antisera, was identified. The sequence of this region was conserved among HPEV1 clinical isolates obtained from Finland and the United States. Antiserum against this peptide region showed neutralizing activity against HPEV1 in cell culture. Because the C-terminal region of HPEV1 VP1 contains a functional RGD motif, the antigenicity of this region was also tested. By using the corresponding peptide antiserum, neutralization of HPEV1 was observed. Cross-neutralization between HPEV1 and coxsackievirus A9, an enterovirus with a similar RGD motif in VP1, was also detected.


Subject(s)
Picornaviridae/immunology , Amino Acid Sequence , Animals , Capsid/chemistry , Capsid Proteins , Epitopes , Humans , Immunoblotting , Immunoenzyme Techniques , Mice , Molecular Sequence Data , Neutralization Tests , Oligopeptides , Rabbits
11.
Biochem J ; 349(Pt 1): 77-84, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10861213

ABSTRACT

Inheritance of the apolipoprotein E (apoE) epsilon 4 allele is a risk factor for late-onset Alzheimer's disease (AD). Biochemically apoE is present in AD plaques and neurofibrillary tangles of the AD brain. There is a high avidity and specific binding of apoE and the amyloid beta-peptide (A beta). In addition to AD apoE is also present in many other cerebral and systemic amyloidoses, Down's syndrome and prion diseases but the pathophysiological basis for its presence is still unknown. In the present study we have compared the interaction of apoE with A beta, the gelsolin-derived amyloid fragment AGel(183-210) and the amyloidogenic prion fragments PrP(109-122) and PrP(109-141). We show that, similar to A beta, also AGel and PrP fragments can form a complex with apoE, and that the interaction between apoE and the amyloidogenic protein fragments is mediated through the same binding site on apoE. We also show that apoE increases the thioflavin-T fluorescence of PrP and AGel and that apoE influences the content of beta-sheet conformation of these amyloidogenic fragments. Our results indicate that amyloids and amyloidogenic prion fragments share a similar structural motif, which is recognized by apoE, possibly through a single binding site, and that this motif is also responsible for the amyloidogenicity of these fragments.


Subject(s)
Amyloid beta-Peptides/chemistry , Apolipoproteins E/chemistry , Peptides/metabolism , Amino Acid Motifs , Amyloidosis/metabolism , Binding Sites , Binding, Competitive , Blotting, Western , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Protein Binding , Protein Conformation , Protein Structure, Secondary , Spectrometry, Fluorescence , Surface Plasmon Resonance
12.
J Clin Microbiol ; 37(12): 3952-6, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10565913

ABSTRACT

The major capsid protein VP2 of human parvovirus B19, when studied in a denatured form exhibiting linear epitopes, is recognized exclusively by immunoglobulin G (IgG) antibodies of patients with acute or recent B19 infection. By contrast, conformational epitopes of VP2 are recognized both by IgG of the acute phase and by IgG of past immunity. In order to localize the VP2 linear epitope(s) specific for acute-phase IgG, the entire B19 capsid protein sequence was mapped by peptide scanning using well-characterized acute-phase and control sera. A unique heptapeptide epitope showing strong and selective reactivity with the acute-phase IgG was detected and characterized. By using this linear epitope (VP2 amino acids 344 to 350) and virus-like particles exhibiting conformational VP2 epitopes, an innovative approach, second-generation epitope-typing enzyme immunoassay, was set up for improved diagnosis of primary infections by human parvovirus B19.


Subject(s)
Capsid Proteins , Capsid/immunology , Epitope Mapping , Immunodominant Epitopes/immunology , Oligopeptides/immunology , Parvoviridae Infections/diagnosis , Parvovirus B19, Human/immunology , Acute Disease , Antibodies, Viral/blood , Antigens, Viral/chemistry , Antigens, Viral/immunology , Capsid/chemistry , Humans , Immunodominant Epitopes/chemistry , Immunoenzyme Techniques , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Parvoviridae Infections/virology
13.
Virology ; 262(2): 321-32, 1999 Sep 30.
Article in English | MEDLINE | ID: mdl-10502511

ABSTRACT

We selected peptide ligands mimicking the surface structure of discontinuous binding sites of Puumala hantavirus-neutralizing monoclonal antibodies from a random 18-amino acid peptide library containing a disulfide bridge in a fixed position and displayed on a filamentous phage. The varying of selection conditions, either by shortening of the association time or by competitive elution with antigen, was crucial for the selection of peptide inserts that could be aligned with the primary sequences of the envelope glycoproteins G1 and G2. Correspondingly, when the envelope glycoprotein sequences were synthesized as overlapping peptides as spots on membrane, the same site in primary structure was found as with phage display, which corroborates the use of the two methods in mapping of conformational epitopes. Also, epitopes reactive with early-phase sera from Puumala virus infection were defined with the pepspot assay in the amino-terminal region of G1. Similarities of the selected phage clones to a monoclonal antibody-escape mutant site and to a linear early-phase epitope were found.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Orthohantavirus/chemistry , Peptide Library , Viral Envelope Proteins/immunology , Amino Acid Sequence , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , Binding, Competitive , Cloning, Molecular , Disulfides/chemistry , Disulfides/metabolism , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Orthohantavirus/genetics , Orthohantavirus/immunology , Hantavirus Infections/blood , Hantavirus Infections/immunology , Hantavirus Infections/virology , Humans , Inovirus/genetics , Molecular Mimicry , Molecular Sequence Data , Neutralization Tests , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Protein Conformation , Sequence Alignment , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics
14.
FEBS Lett ; 449(2-3): 187-90, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10338129

ABSTRACT

Temporin A is a small, basic, highly hydrophobic, antibacterial peptide found in the skin of the European red frog, Rana temporaria. It was synthesized twice by the FastMoc solid phase method using amino acids protected at the N(alpha)-position with either 9-fluorenylmethoxycarbonyl or 2-(4-nitrophenylsulfonyl)ethoxycarbonyl. The syntheses of temporin A demonstrates the difference between 2-(4-nitrophenylsulfonyl)ethoxycarbonyl and 9-fluorenylmethoxycarbonyl amino acids. The purified peptide showed also antibacterial activity against clinically important gram-positive bacteria. It was found to have a moderately good activity against both methicillin resistant and sensitive strains of Staphylococcus aureus, but a weaker activity against vancomycin resistant strains of Enterococcus faecium.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecium/drug effects , Peptides/pharmacology , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/isolation & purification , Peptides/chemical synthesis , Peptides/isolation & purification , Rana temporaria
15.
Virology ; 240(2): 202-12, 1998 Jan 20.
Article in English | MEDLINE | ID: mdl-9454693

ABSTRACT

Antigenic analysis of coxsackievirus A9 (CAV9) was carried out by using a peptide scanning method. Immunogenic regions in the capsid proteins VP1, VP2, and VP3 were recognized by antibodies in the sera of virus-immunized rabbits. The peptide sequences were scanned using a 12-amino-acid window and three-residue shift. Three immunogenic regions, located in the N- and C-terminal parts of VP1 and in the N-terminus of VP3, were identified. Trypsin treatment of the virus, known to cleave off the C-terminus of VP1 containing a functional RGD motif, completely abolished the reactivity against this region but did not have any other significant effect on antigenicity. In further studies, it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sides of the motif. New antigenic sites emerged after heat treatment of CAV9 at 56 or 100 degrees C prior to immunization; in particular, loop structures between beta strands in VP2 exhibited increased immunogenicity. New antigenic sites in VP1 and VP3 also appeared after the treatments. In spite of the markedly altered reactivity in peptide scanning, the virus treated at 56 degrees C elicited high titers of neutralizing antibodies. To reveal cross-reactive antigenic sites, antisera raised against coxsackievirus B3 and echovirus 11 were also tested. The cross-reactive antigenic sites were located mainly in the N-terminal parts of VP1 and VP3.


Subject(s)
Antibodies, Viral/immunology , Capsid/immunology , Enterovirus/immunology , Epitopes/isolation & purification , Animals , Cross Reactions , Epitopes/immunology , Peptides/immunology , Protein Structure, Tertiary , Rabbits
16.
Virus Res ; 51(1): 1-7, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9381791

ABSTRACT

Sequence of the Puumala virus (PUU) Sotkamo strain L segment is provided, completing the total genome of this prototype PUU virus strain. The L segment is 6530 nucleotides long and it can encode 2156 amino-acids-long L protein, RNA-dependent RNA polymerase. The strain Sotkamo, originally isolated in Finland, showed for the L genome segment nucleotide (84.6%) and amino acid (97.3%) homology to a previously sequenced PUU Russian isolate, strain Bashkiria/CG1820 (B1820) and the L genome segment appeared to be at least as conserved as the S segment. Phylogenetic analysis based on the S, M and L segment sequences proposes that the three viral genes have a similar evolutionary history with no evidence for genome segment reassortment. Precise sequencing of the L segment termini demonstrated that the Puumala strains differ from the conserved sequences of the other hantaviruses at two positions.


Subject(s)
Genome, Viral , Orthohantavirus/genetics , Amino Acid Sequence , DNA, Complementary/analysis , DNA, Complementary/chemistry , DNA-Directed RNA Polymerases/genetics , Orthohantavirus/chemistry , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, RNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Viral Proteins/genetics
17.
J Virol ; 71(5): 3879-85, 1997 May.
Article in English | MEDLINE | ID: mdl-9094664

ABSTRACT

We have selected ligands for Puumala hantavirus, the causative agent of nephropathia epidemica, from a seven-amino-acid peptide library flanked by cysteines and displayed on a filamentous phage. To direct the selection to areas on the virus particle which are essential for infection, phages were competitively eluted with neutralizing monoclonal antibodies specific for the viral glycoproteins. The selected phage populations were specific for the same sites as the antibodies and mimicked their functions. The peptide insert, CHWMFSPWC, when displayed on the phages, completely inhibited Puumala virus infection in cell culture at the same effective concentration as the eluting antibody specific for envelope glycoprotein G2. The binding of the phage clones to the virus and inhibition of infection were not necessarily coincident; Pro-6 was critical for virus inhibition, while consensus residues Trp-2 and Phe-4 were essential for binding. The strategy described can be applied to any virus for production of molecules mimicking the effect of neutralizing antibodies.


Subject(s)
Antibodies, Viral/immunology , Bacteriophages/immunology , Oligopeptides/immunology , Orthohantavirus/immunology , Animals , Antibodies, Monoclonal/immunology , Chlorocebus aethiops , Neutralization Tests , Vero Cells , Viral Envelope Proteins/immunology
18.
J Virol Methods ; 62(1): 87-92, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8910652

ABSTRACT

A novel enzyme-linked immunosorbent assay, BLA IgM-ELISA, based on baculovirus-expressed Puumala (PUU) virus nucleocapsid protein, was developed for rapid diagnosis of nephropathia epidemica. The recombinant antigen (bac-PUU-N) was purified to homogeneity by HPLC and conjugated to biotin. The biotin-streptavidin system, in combination with the mu-capture technique, rendered the BLA IgM-ELISA a sensitivity similar to or higher than that of PUU virus IgM mu-capture ELISA based on native antigen. The assay was shown to be highly specific when evaluated using a panel of 160 patient and control sera.


Subject(s)
Antibodies, Viral/analysis , Antigens, Viral/immunology , Capsid/immunology , Enzyme-Linked Immunosorbent Assay/methods , Hantavirus Infections/diagnosis , Orthohantavirus/immunology , Biotin , Hantavirus Infections/immunology , Immunoglobulin M/analysis , Recombinant Proteins/immunology
19.
Virology ; 216(2): 397-406, 1996 Feb 15.
Article in English | MEDLINE | ID: mdl-8607269

ABSTRACT

B-cell epitopes in the nucleocapsid protein (N) of Puumala (PUU) virus were investigated by use of truncated recombinant proteins and overlapping peptides. Six of seven epitopes, recognized by bank vole monoclonal antibodies, were localized within the amino-terminal region of the protein (aa 1-79). Polyclonal antibodies from wild-trapped or experimentally infected bank voles identified epitopes located over the entire protein. Antibody end-point titers to different N fragments indicated that the amino-terminal region is the major antigenic target in PUU virus-infected bank voles. To investigate the role of PUU virus N in protective immunity, we analyzed the immunogenicity of truncated recombinant N and developed an animal model based on colonized bank voles. No PUU virus N antigen, nor any glycoprotein-specific antibodies, could be detected after virus challenge in animals immunized with an amino-terminal fragment (aa 1-118), a fragment covering two thirds of the animals immunized with shorter N fragments displayed either N antigen, or glycoprotein-specific antibodies, suggestive of partial protection. Prechallenge sera from all groups of immunized animals were found negative or only weakly positive for neutralizing antibodies when assayed by focus reduction neutralization test, which indicated an important role for cell-mediated immunity in protection.


Subject(s)
Capsid/immunology , Epitopes, B-Lymphocyte/immunology , Hantavirus Infections/prevention & control , Orthohantavirus/immunology , Viral Core Proteins/immunology , Viral Vaccines/immunology , Animals , Arvicolinae , Base Sequence , Capsid/genetics , Cell Line , Chlorocebus aethiops , DNA Primers , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Gene Expression , Hantavirus Infections/immunology , Humans , Molecular Sequence Data , Peptide Fragments/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spodoptera , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vero Cells , Viral Core Proteins/genetics , Viral Vaccines/genetics
20.
J Clin Microbiol ; 34(1): 119-25, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8748286

ABSTRACT

Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome. Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein. In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein. The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography. In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells. An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen. Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized. Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.


Subject(s)
Antigens, Viral , Capsid/genetics , Capsid/immunology , Hantavirus Infections/diagnosis , Orthohantavirus/genetics , Orthohantavirus/immunology , Viral Core Proteins/genetics , Viral Core Proteins/immunology , Animals , Antibodies, Monoclonal , Antibodies, Viral/blood , Antigens, Viral/genetics , Baculoviridae/genetics , Cell Line , Evaluation Studies as Topic , Gene Expression , Hantavirus Infections/immunology , Hantavirus Infections/virology , Humans , Immunoassay/methods , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Spodoptera
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