Serological evaluation of possible exposure to Ljungan virus and related parechovirus in autoimmune (type 1) diabetes in children.

Exposure to Ljungan virus (LV) is implicated in the risk of autoimmune (type 1) diabetes but possible contribution by other parechoviruses is not ruled out. The aim was to compare children diagnosed with type 1 diabetes in 2005-2011 (n = 69) with healthy controls (n = 294), all from the Jämtland County in Sweden, using an exploratory suspension multiplex immunoassay for IgM and IgG against 26 peptides of LV, human parechoviruses (HPeV), Aichi virus and poliovirus in relation to a radiobinding assay (RBA) for antibodies against LV and InfluenzaA/H1N1pdm09. Islet autoantibodies and HLA-DQ genotypes were also determined. 1) All five LV-peptide antibodies correlated to each other (P < 0.001) in the suspension multiplex IgM- and IgG-antibody assay; 2) The LV-VP1_31-60-IgG correlated with insulin autoantibodies alone (P = 0.007) and in combination with HLA-DQ8 overall (P = 0.022) as well as with HLA-DQ 8/8 and 8/X subjects (P = 0.013); 3) RBA detected LV antibodies correlated with young age at diagnosis (P < 0.001) and with insulin autoantibodies (P < 0.001) especially in young HLA-DQ8 subjects (P = 0.004); 4) LV-peptide-VP1_31-60-IgG correlated to RBA LV antibodies (P = 0.009); 5) HPeV3-peptide-IgM and -IgG showed inter-peptide correlations (P < 0.001) but only HPeV3-VP1_1-30-IgG (P < 0.001) and VP1_95-124-IgG (P = 0.009) were related to RBA LV antibodies without relation to insulin autoantibody positivity (P = 0.072 and P = 0.486, respectively). Both exploratory suspension multiplex IgG to LV-peptide VP1_31-60 and RBA detected LV antibodies correlated with insulin autoantibodies and HLA-DQ8 suggesting possible role in type 1 diabetes. It remains to be determined if cross-reactivity or concomitant exposure to LV and HPeV3 contributes to the seroprevalence.

Generation, application and quantification of clamp-BioShuttle carriers for plasmid delivery into nuclei of prostate cancer cells.

This paper presents the BioShuttle platform as a delivery vehicle for transfer of contrast agents and genetic material into target cells, which can be followed by activation of the BioShuttle inside the target cell. Here, we present a transporter system and summarize the findings on transporter use in vivo and in vitro. The results here are limited to examples where cargoes (drugs, genetically active materials or contrast agents) are covalently associated with the transporter module. A further example, in which the cargo is non-covalently attached to the BioShuttle, is also discussed. Finally, attempts have been made to solve some of the issues surrounding the efficiency of transfer of therapeutic or diagnostic agents and their later activity in the cell.

Development and characterization of drug delivery systems for targeting mammalian cells and tissues: a review.

Recent advances in the development of diagnostics and therapeutics in the fields of recombinant biochemistry, solid phase peptide synthesis as well as in galenical research have resulted in highly specific and efficient components. Presently, millions of patients can profit from these new therapeutic modalities. The application of an effective anti-tumor dose of drugs can lead to marked toxicity in patients. Therefore, safe and efficient possibilities to transport these compounds to the target are of outmost importance. The importance of drug delivery is pivotal in the wide area of pharmacological research. However, until now, this issue is still to be solved. The main goal of every drug delivery system is the delivery of a precise amount of a drug at a pre-programmed rate to the desired location in order to achieve the necessary drug concentration in the targeted organ for effective treatment. The key problem still remains the achievement of curative doses in a pharmacologically active state in the desired target while avoiding side effects. Although respectable advances can be recognized in this field, the currently applied mechanisms for the transport of therapeutic molecules across biological membranes still remain far from being efficient. Helper molecules could improve delivery to desired target sites. Presently, a number of efforts are made and a huge spectrum of biochemical, biological, medical, pharmaceutical and physical possibilities are arising. However, the design and development of successful therapies based on this technology still remains a great challenge.

Molecular imaging: Bridging the gap between neuroradiology and neurohistology.

Historically, in vivo imaging methods have largely relied on imaging gross anatomy. More recently it has become possible to depict biological processes at the cellular and molecular level. These new research methods use magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared optical imaging, scintigraphy, and autoradiography in vivo and in vitro. Of primary interest is the development of methods using MRI and PET with which the progress of gene therapy in glioblastoma (herpes simplex virus-thymidine kinase) and Parkinson's disease can be monitored and graphically displayed. The distribution of serotonin receptors in the human brain and the duration of serotonin-receptor antagonist binding can be assessed by PET. With PET, it is possible to localize neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. MR tracking of transplanted oligodendrocyte progenitors is feasible for determining the extent of remyelinization in myelin-deficient rats. Stroke therapy in adult rats with subventricular zone cells can be monitored by MRI. Transgene expression (beta-galactosidase, tyrosinase, engineered transferrin receptor) can also be visualized using MRI. Macrophages can be marked with certain iron-containing contrast agents which, through accumulation at the margins of glioblastomas, ameliorate the visual demarcation in MRI. The use of near-infrared optical imaging techniques to visualize matrix-metalloproteinases and cathepsin B can improve the assessment of tumor aggressiveness and angiogenesis-inhibitory therapy. Apoptosis could be detected using near-infrared optical imaging representation of caspase 3 activity and annexin B. This review demonstrates the need for neurohistological research if further progress is to be made in the emerging but burgeoning field of molecular imaging.

Synthesis and application of functional peptides as cell nucleus-directed molecules in the treatment of malignant diseases.

The unique functions of biomolecules, including transport across biological membranes (e.g. the cell membrane, the nuclear envelope), modulation of protein function, gene transcription, reconstitution of the malignant transformation, and viral, bacterial and fungal activities underlie a high pharmaceutical potential. The development of combinatorial functional peptide modules in this important area has been slow, in contrast to the rapid development in the synthesis of small biopolymers. The conjugation of a short transmembrane transport peptide module with a cell nucleus address peptide module and with any substance is attractive for preparation of BioShuttle-based peptides because of the well-established automated synthesis of peptides. Variation of the different functional modules for drug targeting and the choice of substances can be combined to create novel bioconjugates with unique properties. This article provides an overview of previous work on the BioShuttle technology and outlines the promising use of this approach in combinatorial peptide synthesis and drug discovery.

Analysis of protein tyrosine phosphorylation by nanoelectrospray ionization high-resolution tandem mass spectrometry and tyrosine-targeted product ion scanning.

A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.

High-throughput peptide synthesis and peptide purification strategy at the low micromol-scale using the 96-well format.

The increasing demand for short- and medium-sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high-throughput synthesis and purification techniques at the low-micromol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96-well format. First, a micromol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96-reaction vessel block employing standard Fmoc/tBu-chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix-assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed-phase solid-phase extraction (IP-RP-SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the micromol-scale synthesis in the 96-well format with purification by IP-RP-SPE on a 96-well micro-extraction plate format. This simple, fast and parallel approach was tested on 12-mer and 15-mer peptides to map epitopes of T- and B-cell clones, respectively. Approximately 80% of all peptides were obtained at purities > 90% without purification by RP-HPLC. In summary, this novel approach has several advantages: (i) the micromol-scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP-HPLC column was not contaminated with hydrophobic impurities.

Peptide mimics of the vesicular stomatitis virus G-protein transmembrane segment drive membrane fusion in vitro.

The efficiency of cell-cell fusion mediated by heterologously expressed vesicular stomatitis virus G-protein has previously been shown to be affected by mutating its transmembrane segment. Here, we show that a synthetic peptide modeled after this transmembrane segment drives liposome-liposome fusion. Addition of millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides suggesting that Ca(2+)-mediated liposome aggregation supports the activity of the peptide. Peptide-driven fusion was suppressed by lysolipid, an established inhibitor of natural membrane fusion, and involved inner and outer leaflets of the liposomal bilayer. Thus, transmembrane segment peptide-driven liposome fusion exhibits important hallmarks characteristic of natural membrane fusion. Importantly, the mutations previously shown to attenuate the function of full-length G-protein in cell-cell fusion also attenuated the fusogenicity of the peptide, albeit in a less pronounced fashion. Therefore, the function of the peptide mimic is dependent on its primary structure, similar to full-length G-protein. Together, our data suggest that the G-protein transmembrane segment is an autonomous functional domain. We propose that it acts at a late step in membrane fusion elicited by vesicular stomatitis virus.

Sequence evolution and cross-reactive antibody responses to hypervariable region 1 in acute hepatitis C virus infection.

Hepatitis C virus (HCV) infection may result in acute resolving or chronic infection. Patients that clear the infection have a more vigorous cellular immune response and an early humoral response to the hypervariable region 1 (HVR1) of the E2 envelope protein. To analyse further the properties of the early anti-HVR1 response, cross-reactivity of anti-HVR1 responses was assessed in five patients with acute HCV infection, who were infected by the same virus strain during a nosocomial outbreak. The sequence evolution of HVR1 was examined in sequential serum samples up to 37 months post infection. Peptides were synthesised corresponding to the obtained HVR1 sequences and unrelated HVR1 sequences, and antibody reactivity to the peptides in sequential sera was investigated by ELISA. The results suggest an association between specific gaps in humoral immunity and the HVR1 sequence evolution during early infection. Possible interpretations of this phenomenon include immune escape mechanisms or suppression of specific anti-HVR1 antibodies.

Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry.

Loss of phosphoric acid is the most effective fragmentation reaction of pSer- and pThr-containing phosphopeptides of small size (up to 10-15 residues) in low-energy collision-induced dissociation. Therefore, tandem mass spectrometry with neutral loss scanning was evaluated for its utility to analyze protein phosphorylation using protein kinase A (PKA) catalytic subunit, which is phosphorylated at Thr197 and Ser338, as an example. Analysis of tryptic digests of phosphoproteins by tandem mass spectrometry with scanning for neutral loss of phosphoric acid resulted in spectra with poor signal-to-noise ratio, mainly because of the large size of the phosphopeptides formed (>2 kDa). This unfavorable size was caused by the distribution of tryptic cleavage sites in PKA and by interference of phosphorylation with tryptic cleavage. To generate a set of smaller peptide fragments, digestion was performed using the low-specificity protease elastase. Analysis of the total elastase digest with neutral loss scanning resulted in observation of a set of partially overlapping phosphopeptides with high abundance, providing a complete coverage of PKA phosphorylation sites. The peptide size generated by elastase (0.5-1.5 kDa) is ideally suited for this scan mode, which was found to provide the highest specificity for detection of singly charged phosphopeptides (neutral loss of 98). Identification of the PKA phosphorylation sites was performed by mass spectrometric sequencing of the elastase-derived phosphopeptides, which provided highly informative product ion spectra.

Influence of myristoylation, phosphorylation, and deamidation on the structural behavior of the N-terminus of the catalytic subunit of cAMP-dependent protein kinase.

A number of isoenzymes of the catalytic subunit of cAMP-dependent protein kinase arise through posttranslational modifications of the enzyme outside the catalytic domain; the biological significance of these is not yet fully clear. A clustering of sites for such modification exists at the N-terminus of the protein, where myristoylation (of Gly1), phosphorylation (at Ser10), and deamidation of Asn2 have been observed. As the first two are known to govern membrane binding and thus subcellular compartmentalization in some proteins, it was of interest to see whether the local structure of the N-terminus was being influenced by one or more of these modifications. A series of synthetic peptides mimicing the 16 N-terminal residues of the catalytic subunit Calpha was produced covering the full range of possible modifications, singly and in combination, and tested for possible effects on local structure by measuring the circular dichroism under varying polarity. It was found that myristoylation and phosphorylation modify the structure in this region in opposite ways and in a manner designed to amplify the action of a potential myristoyl/electrostatic switch. To what extent deamidation of Asn2 may oppose a potential membrane binding is unknown. Deamidation, however, had no effect on the structure of the peptide either alone or in combination with acylation and/or phosphorylation, suggesting that the change of the nuclear/cytoplasmic disribution in cells caused by deamidation [Pepperkok et al. (2000) J. Cell Biol. 148, 715-726] is due to a more complex signaling mechanism. The structural implications of the data are discussed.

Increased immunoglobulin G, but not M, binding to endogenous retroviral antigens in HIV-1 infected persons.

The modes of interaction between products of human endogenous retroviral (HERV) sequences and the immune system are largely unknown. In HIV infected persons, an exogenous retrovirus adds further complexity to the situation. Therefore, 14 synthetic peptides with sequences derived from conserved regions of various endogenous retroviruses (ERVs) and from related exogenous retroviruses were used to search for IgG and IgM antibodies that bind to such antigens in 15 HIV-1 seropositive and 17 seronegative immunosuppressed patients. IgG binding to three peptides, namely, the C-terminal half of murine leukemia virus (MLV) capsid protein, the conserved portion of HERV-H transmembrane protein, and the Pol region of human mouse mammary tumor virus (MMTV)-like (HML3) sequence, was observed in both groups. Binding was, however, more frequent and more firm in HIV-1 positive samples (P<0.0001, Wilcoxon rank sum test). IgM binding to the same peptides showed no significant differentiation between the two groups of patients. Binding to both immunoglobulin isotypes was sometimes variable over time in both groups. No correlation of either IgG or IgM peptide binding with progression to AIDS in HIV-1 infected individuals was observed. Inhibition studies using analogous endogenous and exogenous retroviral peptides, including HIV-1, demonstrated specificity of the IgG antibodies for a narrow range of MLV- and MMTV-like retroviral antigens, and excluded cross-reactivity of antibodies to HIV-1 as a cause of these observations. Thus, unlike IgG, IgM binding to retroviral antigens was ubiquitous. It is suggested that anti-HERV IgM belong to a class of natural antibodies and might serve as primers in the mediation of humoral immune responses to more or less related exogenous retroviruses. Increased IgG binding in HIV-1 infected individuals could result from such priming, or reflect higher HERV antigen expression.

Structural prerequisites for intersubtype B and D antigenicity of the third variable envelope region (V3) of human immunodeficiency virus type 1.

To elucidate the structural requirements for intersubtype antigenicity of human immunodeficiency virus type 1 (HIV-1) third variable envelope region (V3), synthetic peptides were used in enzyme immunoassays (EIAs) with serum samples from persons with proven or probable subtype B and D infections. Mathematical analyses of results from EIAs with singly substituted V3 peptides revealed important residues determining overall N-terminal V3 peptide antigenicity. This information was used to design V3 immunogens, rabbit antiserum to which were tested in EIA and for in vitro neutralization of molecular clones of HIV-1(MN) and HIV-1(MAL). Intersubtype-reactive epitopes were distributed toward the N-terminal half of the V3 loop. Lysine at position 310, arginine at position 311, and isoleucine at position 314, all derived from the MN primary sequence, were major determinants of intersubtype V3 antigenicity. Combinations of residues that enhanced antigenicity often contained lysine at position 310. Threonine at position 308 was common in the least advantageous combinations. V3 immunogens modified to achieve optimal antigenicity induced antiserum with augmented cross-neutralization of virus from MAL and MN molecular clones, suggesting one approach to subunit vaccine development.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

The amino terminus of PKA catalytic subunit--a site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes.

Conserved deamidation of PKA catalytic subunit isozymes Calpha and Cbeta--more than 25% at Asn2 in vivo in both cases--has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide.

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.

Identification of structural elements of subunit beta of human protein kinase CK2 participating in tight physical alpha-beta intersubunit contacts directly adjacent to a surface-oriented region.

Sites essential for tight physical intersubunit interaction in protein kinase CK2, a tetramer composed of two catalytic (alpha and/or alpha') and two regulatory subunits (beta), have been assigned to the C-terminal part of subunit beta. Mutational analysis suggests region 171-181 of beta to be one of these but this is not consistent with the observation of coprecipitation of catalytic subunits by antibodies directed specifically to this beta segment which indicates that this region is accessible to antibodies even if the beta subunit is associated with the alpha subunit. In an attempt to clarify the apparent contradiction, we have subdivided beta-(155-181)-peptide, which includes the fragment of beta and that both binds to catalytic subunits and stimulates kinase activity, into six more or less overlapping peptides with a length of 9-16 amino acid residues and performed peptide competition and a subunit binding assays. The kinase-stimulating effect of beta-(155-181)-peptide was counteracted significantly by beta-(162-175)-peptide while the others had less or no effect. In an ELISA assay with the peptides covalently bound to wells via a C10 spacer arm, binding of a occurred besides beta-(155-181)-peptide only with beta-(162-175)-peptide and beta-(165-175)-peptide. The data provide the first evidence that the contact and the stimulation competences of subunit beta represent separable functions and suggest further that a tight physical contact to catalytic subunits is located at position 162-175 consisting of a hydrophobic stretch (position 162-171) and a hydrophilic binding motif, PEY (position 172-174), with the adjoining downstream part allowing for surface exposure and antibody binding. The presence of several Pro residues within that region might relate to the structural basis for both the alpha-beta interaction and the surface orientation.

Two domains of the Borna disease virus p40 protein are required for interaction with the p23 protein.

Borna disease virus (BDV) has five major open reading frames, which encode the proteins p40, p23, gp18, p57 and p190. By analogy with other negative-strand RNA viruses, p40 is a putative nucleoprotein and p23 is a putative phosphoprotein. These proteins are known to form complexes with each other and with the polymerase protein in other viruses. In this paper, it is shown that BDV p40 and p23 can form complexes with each other in infected cells. Furthermore, the amino acids of p40 that are necessary for formation of this complex have been mapped.

Selective antibody reactivity with peptides from human endogenous retroviruses and nonviral poly(amino acids) in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate antibody responses to a broad panel of peptides derived from human endogenous retroviruses (HERVs) among unselected patients with systemic lupus erythematosus (SLE). METHODS: In sera obtained from 69 patients with SLE and healthy blood donors, immunoassay was used to measure levels of antibody against synthetic peptides derived from HERVs, exogenous retroviruses, and nonviral poly(amino acids). RESULTS: Measurement by immunoassay revealed increased frequencies of antiretroviral antibodies against 2 peptides derived from the env gene of the type C-like class, which includes ERV-9 and HERV-H, and against 2 peptides from the gag region of human T lymphotropic virus type I-related endogenous sequence 1, in patients with SLE. Antibodies to 2 nonviral peptides, polyhistidine and polyproline, were also overrepresented in patient sera. In 1 patient, longitudinal data obtained over a period of 12 years indicated that the concentrations of certain antiretroviral antibodies varied according to disease activity. CONCLUSION: Reactivity to certain type C HERV-derived antigens was found among patients with SLE. This reactivity could be explained by increased exposure to cross-reactive epitopes from essentially complete type C HERVs.