DE19908766C2 - Use of synthetic Vpr peptides of the human immunodeficiency virus type 1 (HIV-1) for the development of therapeutic and diagnostic reagents - Google Patents

Abstract

The invention relates to new, synthetically produced Vpr proteins, their biological activities and their antigenic and physicochemical properties which are similar or identical to those of viral Vpr expressed by natural, HIV-infected cells. This describes a method with which sVpr proteins are made available in practically unlimited quantities, with high purity and under economically justifiable conditions for the development of novel antiviral reagents and serological test systems. DOLLAR A areas of application are - in addition to basic HIV research and the structural analysis of HIV systems - peptide chemistry, the development of test systems for screening potential Vpr antagonists, the establishment of cell culture and animal models for the investigation of the pathomechanisms of Vpr, the development of Serological test methods, in particular a Vpr antigen ELISA and the in vitro assembly of novel vectors for use in gene transfer methods in gene therapy.

Description

The invention relates to the use of synthetic peptides of the regulatory virus protein R (Vpr) of Human Immunodeficiency Virus Type 1 (HIV-1) for the purpose of establishing novel ones Test methods in the serological diagnosis and monitoring of HIV-1 infected patients who Clarification of the molecular structure and mode of action of this both for virus replication and also for the viral pathomechanisms essential virus protein. Based on that gained knowledge about its molecular and physicochemical properties with the use of novel synthetic peptides that are either part or total sequences of HIV-1 Vpr, principle solutions for novel antiviral strategies for use in the therapy and treatment of HIV infections. In addition, the novel Products in the form of Vpr peptides for the establishment of gene transfer methods in gene therapy applied.

Characteristic of the known prior art

• 1.1.1. introduction
  • 1.1.1.1. Genomic organization of retroviruses
  • 2. 1.1.2. HIV replication cycle
  • 3. 1.1.3. Importance of accessory viral gene products in the replication cycle
  • 4. 1.1.4. The accessory regulatory protein Vpr
• 2. 1.2. Analysis of the biological functions of Vpr
  • 1. 1.2.1. Importance of Vpr in vivo for the pathogenesis of HIV infections
  • 2. 1.2.2. Vpr supports virus replication in vitro
  • 3. 1.2.3. Vpr induces cell differentiation and cell cycle arrest
  • 4. 1.2.4. Nuclear transport and function (s) of Vpr in the cell nucleus
  • 5. 1.2.5. Interaction of Vpr with cellular factors
  • 6. 1.2.6. Vpr regulates the effects of glucocorticoid hormones
  • 7. 1.2.7. Incorporation of Vpr into virus particles
• 3. 1.3. Biochemical and structural characterization of Vpr
• 4. 1.4. Chemical synthesis and activities of Vpr peptides
• 5. 1.5. Expression and Activities of Recombinant Vpr
  • 1.5.1.1. Expression of Vpr in insect cells
  • 2. 1.5.2. Expression of Vpr in Escherichia (E.) coli
  • 3. 1.5.3. Patent literature

1.1. introduction

In the past two decades, the pandemic of acquired immune deficiency syndrome (Aquired Immunodeficiency Syndrome, AIDS) Millions of people infected with HIV with one face insidious, multisystemic and ultimately incurable disease. Notwithstanding the relatively extensive detailed knowledge of the genomic organization, the regulation of Viral gene expression as well as the biological functions of most HIV proteins is well known the replication and spread of the pathogen in vivo as well as via the concrete Pathogenicity mechanisms are still incomplete. New hope for the identification of previously unknown factors that trigger the clinical progression of the disease and / or promote, arises with the evaluation of "long term survivors" of an HIV infection. One focus is the identification and characterization of virus-specific Pathogenicity factors. Of particular interest here are the non-structural proteins of the virus, which exercise regulatory functions in the replication cycle of HIV (bibliography on the status of Technology behind the examples).

1.1.1. Genomic organization of retroviruses

The retrovirus family, which includes HIV, belongs to the large group of eukaryotic retrotransposable elements (Doolittle et al., 1989, 1990). These are characterized by the ability to convert RNA genomes into DNA using the reverse transcriptase enzyme Intermediate rewrite (Doolittle et al., 1989). Retroviruses come in five subfamilies broken down: (i) spuma viruses; (ii) Mammalian C-type oncoviruses; (iii) BLV (Bovine Leukemia Virus) / HTLV (Human T-Cell Leukemia Virus) leukemia viruses; (iv) a heterogeneous group from RSV (Rous Sarcoma Virus), A, B and D-type viruses; and (v) lentiviruses (Doolittle et al., 1990; Martin, 1993). As the name suggests, lentiviruses cause long-lasting and mostly incurable Diseases. Lentiviruses replicate primarily in lymphocytes and differentiated macrophages. Replication-competent retroviruses contain at least three characteristic genes: gag (group-specific antigen), pol (polymerase) and env (coat proteins). Except structural and Various retroviruses encode additional, usually small, enzymatically active virus proteins Proteins with regulatory functions. In addition to HIV, the subfamily of lentiviruses include SIV (Simian Immunodeficiency Virus), EIAV (Equine Infectious Anemia Virus), BIV (Bovine Immunodeficiency Virus), FIV (Feline Immunodeficiency Virus) and Visna Virus (Doolittle et al., 1990). HIV in turn is divided into the two subtypes HIV-1 and HIV-2 (Clavel et al., 1987). In a comparable genome organization, HIV-1 is caused by the vpu gene (Strebel et al., 1988; Cohen et al., 1988) and HIV-2 by the vpx gene (Henderson et al., 1988).

1.1.2. HIV replication cycle

The replication cycle of HIV begins with the virus binding to various cell receptors,  among which the CD4 glycoprotein acts as the primary receptor. The virus enters through pH-independent membrane fusion, followed by the "uncoating" of the virus particles in the cytosol. The viral RNA genome is generated by means of enzymatic activities of reverse transcriptase (RT), RNase H and Polymerase rewritten into double-stranded DNA, which is then associated with the Pre-integration complex transported into the cell nucleus and as a provirus genome in chromosomal DNA is incorporated by means of viral integrase. After transcription and translation, Gag / Gag-Pol- Polyproteins as well as coat proteins are transported to the cell membrane, where the assembly of virions he follows. After budding and constriction, virus particles mature through proteolytic processing of the Gag / Gag-Pol polyproteins (for review see Martin, 1993).

1.1.3. Importance of accessory viral gene products in the replication cycle of HIV-1

In addition to the prototype retrovirus genes gag, pol and env, HIV-1 encodes other genes: tat and rev are essential for virus replication; Tat is the main transactivator of the virus promoter (Dayton et al., 1986; Fisher et al., 1986) and Rev performs post-transcriptional functions by transporting non-spliced gag / pol as well as single spliced vif, vpr and vpu / env mRNAs from the cell nucleus in regulates the cytoplasm (Malim et al., 1989; for review see Martin, 1993 and Trono, 1995). In contrast, nef, vif, vpr and vpu are not absolutely necessary for virus replication in cell culture and are therefore commonly referred to as "accessory" genes. Although in the past few years The accessory genes of HIV-1 have been studied intensively, the knowledge of their molecular ones Mechanisms only incomplete. Controversial about the extremely high mutation and the resulting high evolutionary rate of retroviruses (especially for HIV) is the observation that deletions of these accessory genes only cause subtle changes in the phenotype in cell cultures and usually do not significantly affect replication of such mutants in vitro. Indeed However, there are increasing indications in the literature that these proteins spread the infection and Determine disease progression of individual viruses in vivo significantly. Furthermore, experiments in demonstrated in vitro that individual of these accessory proteins in certain cell cultures, especially in cultured primary human monocytes / macrophages, an essential function for the Exercise virus replication.

1.1.4. The accessory regulatory protein Vpr

The vpr gene was first introduced in 1987 as a small open reading frame in the human genome Immunodeficiency virus type 1 (HIV-1) described and designated with the letter "R" (Wong-Staal et al., 1987). The corresponding gene product, the approximately 15 kDa Vpr protein, is immunogenic and induces anti-Vpr sero antibodies in the majority of all HIV-1 infected (Wong-Staal et al., 1987). The vpr gene is among all known isolates of HIV-1 and HIV-2 as well as the monkey Immunodeficiency Virus (SIV) conserves and is therefore also called the lentivirus protein Vpr. The 96 amino acid Vpr protein of HIV-1 shows structural and functional similarities  the accessory protein Vpx specific for HIV-2 and SIV (Tristem et al., 1992). It will therefore believed that Vpx was caused by gene duplication of Vpr during the evolution of HIV-2 / SIV viruses arose.

Vpr has various biological activities such as: oligomerization, localization in and Transport of the viral pre-integration complex to the cell nucleus, transcription activation of HIV-1 and heterologous virus promoters, induction of cell differentiation and cell cycle arrest, binding and interaction with various cellular proteins, particularly the glucocorticoid receptor (GR) and enhancement of the effects of steroid hormones (summarized in Emerman, 1996 and Levy et al., 1994).

1.2. Analysis of the biological functions of Vpr 1.2.1. Importance of Vpr in vivo for the pathogenesis of HIV infections

Animal model studies showed, for example, that Vpr is necessary for the development of AIDS-like symptoms in SIV-infected rhesus monkeys, or that point mutations in the vpr gene revert to functional Vpr in vivo as the infection spreads (Lang et al., 1993) . On the other hand, similar studies have shown that AIDS occurred in SIV _mac 239-infected rhesus monkeys regardless of the expression of Vpr and Vpx (Gibbs et al., 1995). Given that Vpx of HIV-2 and SIV was caused by gene duplication of Vpr in the evolution of primitive lentiviruses (Tristem et al., 1992), it would be possible that Vpx could complement the function of Vpr. In fact, the double mutations in vpx and vpr of SIV _mac 239 prevent AIDS progression (Gibbs et al., 1995).

Similar evidence for a function of Vpr in vivo has been provided by molecular and phylogenetic studies of the vpr gene from HIV-1 isolates, obtained from long-term survivor subjects who lived more than 13 years without the occurrence of AIDS similar diseases and with a stable CD4 ⁺ T cell level survived (Wang et al., 1996). Most of the vpr genes showed mutations, especially within the C-terminus coding region. This basic region of Vpr is said to be responsible for Vpr-induced G2 cell cycle arrest (Di Mario et al., 1995). In contrast, no mutations were found in the comparison group of AIDS patients.

1.2.2. Vpr supports virus replication in vitro

Vpr is the only accessory protein that is available in significant amounts and through a specific mechanism is actively incorporated into virus particles (Cohen et al., 1990a, b; Yu et al., 1990). This fact led to the presumption that the effects of Vpr early in HIV Replication cycle takes place immediately after membrane fusion and "uncoating" the penetrating Virus particle. This assumption is based on the fact that Vpr is in the virus particle apparently has no influence on the morphology and infectivity of HIV virions.  

Indeed, in vitro studies have shown that mutations in vpr reduce virus replication and cytopathogenicity of HIV-1, HIV-2 and SIV in primary CD4 ⁺ T lymphocytes and transformed T cell lines (Ogawa et al., 1989; Shibata et al ., 1990a, b; Westerwelt et al., 1992). There are even individual reports that indicate that mutations in vpr of HIV-2 can completely prevent infection of primary monocytes / macrophages (Hattori et al., 1990).

Other studies on a possible use of "anti-Vpr drugs" have shown that vpr is specific antisense constructs containing phosphorothioate deoxynucleotides, HIV-1 virus replication in macrophages (Balotta et al., 1993).

In summary, it can be said that Vpr is not essential for virus replication in most established permanent CD4 ⁺ T cell lines. Furthermore, Vpr has little or no effect on HIV infection of activated, rapidly dividing peripheral blood lymphocytes ("peripheral blood mononuclear cells, PBMC") (Dedera et al., 1989). However, Vpr supports and / or enables virus replication in differentiated, no longer dividing primary human monocytes / macrophages (Bukrinsky et al., 1993; Heinzinger et al., 1994; Westervelt et al., 1992; Hattori et al., 1990; Connor et al., 1995). This fact is justified by the fact that Vpr is necessary for the import of the viral pre-integration complex into the cell nucleus of cells that no longer divide and can thus infect HIV differentiated cells independently of cell mitosis (Heinzinger et al., 1994; Fletcher et al., 1996 ).

1.2.3. Vpr induces cell differentiation and cell cycle arrest

Initial studies on this topic showed that Vpr influences cell growth and cell cycle as well as terminal differentiation processes independently of other viral factors in tumor cell lines (Levy et al., 1993). Subsequent studies clarified that Vpr interferes with the proliferation of cells infected with HIV-1 by blocking the cell cycle in the G2 phase (Di Marzio et al., 1995; He et al., 1995; Re et al., 1995; Rogel et al., 1995). The arrest in the transition from G2 to the mitotic (M) phase immediately after duplication of the chomosomal DNA generally results in cell growth arrest and apoptosis. This phenomenon can explain the general observation that vpr ⁺ viruses generally cannot establish a chronic infection in T cells, whereas vpr-deficient viruses enable long-term cultures of chronically infected cells (Rogel et al., 1995). Vpr itself has no effect on the cytopathic processes of an acute HIV infection.

Vpr-induced cell cycle arrest is believed to be a general cellular Pathway is involved, as this phenomenon occurs not only in human cell lines, but also in not human primate cells and in primitive eukaryotes such as yeast (Planelles et al., 1996; Zhao et al., 1996; Stivahtis et al., 1997).

The effect of Vpr on cell division and differentiation can also be triggered by soluble, extracellular Vpr. In this context, it is noteworthy that Vpr occurs in concentrations of up to 10 ng / ml in the serum as well as in the cerebal spinal liquor of HIV-infected people (Levy et al., 1994). Such a "serum Vpr" can - isolated from peripheral blood of HIV-1 infected subjects - activate latent HIV-1 expression in T cell lines as well as in cultured PBMC. This observation suggested that Vpr regulates HIV latency in vivo. In fact, the level of serum Vpr correlates with the level of p24 antigenemia and the disease progression of individual infected people (Levy et al., 1995). The presence of soluble serum Vpr in peripheral blood and in spinal liquor of HIV-infected people is explained by lysis and by the immunological disintegration of virus particles. Serum Vpr is believed to contribute to the known pathologies associated with HIV infection, including CD4 ⁺ T cell loss and neurological symptoms. Similar to the effect of serum Vpr, recombinant Vpr produced in insect cells in concentrations of 0.1 to 10 ng / ml can stimulate virus replication in acutely as well as latently infected cell lines and primary cells and thereby complement the defect of vpr-deficient viruses in trans ( Levy et al., 1995).

1.2.4. Nuclear transport and function (s) of Vpr in the cell nucleus

The ability of virus replication in non-dividing cells, such as differentiated macrophages, which is specific for lentiviruses essentially depends on the transport of the viral genome in conjunction with the pre-integration complex into the cell nucleus, a process which takes place independently of the breakup of the nuclear membrane during the course of the Mitosis occurs. Part of the HIV pre-integration complex are viral nucleic acids, enzymes such as integrase and reverse transcriptase, the nucleocapsid protein NCp7, the matrix protein p17 ^MA and Vpr. So far, the mechanisms that regulate the nuclear transport of Vpr have not been clarified, especially since Vpr does not have any of the known nuclear localization signals (NLSs). The biological significance of the binding of Vpr with the glucocorticoid receptor type II (GR-II) (Refaeli et al., 1995) and the mechanisms of Vpr-induced G2 arrest are still not understood. It remains to be clarified whether the non-specific activation of HIV-1 and other viral promoters of HTLV-1, EBV and CMV (Cohen et al., 1990) are important for these functions of Vpr in the cell nucleus. Recent studies have shown that a 20 amino acid long peptide from the negatively charged N-terminal domain of Vpr is sufficient to effect the transport of bovine serum albumin into the cell nucleus (Karni et al., 1998).

In the cell nucleus, complexes consisting of Vpr and NCp7 should activate phosphatase 2A ₀ and thereby trigger G2 arrest (Tung et al., 1998). The protein phosphatase 2A ₀ acts as an inhibitor in the transition from G _O to the M phase in the process of cell division and also influences various other cellular processes.

1.2.5. Interaction of Vpr with cellular factors

Since Vpr as a component of the pre-integration complex influences elementary processes of the cell in the interest of virus replication, it could be assumed that Vpr should make specific connections with cellular factors. In fact, an interaction of Vpr with an approximately 200 kDa protein and with a 41 kDa cellular protein as a complex with the human glucocorticoid receptor type II (GR-II) was observed (Zhao et al., 1994; Refaeli et al., 1995) . Vpr also suppresses T cell receptor-mediated apoptosis and T cell activation (activation of lymphokine expression IL-2, IL-10, IL-12, TNF-alpha and IL-4) by influencing NF- _k B activity (Ayyavoo et al ., 1997). In the same study, it was shown that both Vpr and glucocorticoids such as dexamethasone and hydrocortisone block cell division equally, while the GR antagonist RU 486 canceled the Vpr effect. These (Ayyavoo et al., 1997) and other studies (Rafaeli et al., 1995) support the hypothesis that the Vpr-mediated G2 arrest takes place in whole or in part via the GR-mediated pathway.

Furthermore, Vpr with the second UBA (ubiquitin associated) domain of human DNA Repair protein HHR23A enter into a specific interaction, which is a three-helix Bundle structure forms (Diekmann et al., 1998). This interaction in turn supports the Vpr- induced G2 cell cycle arrest (Withers-Ward et al., 1997). However, there is direct evidence for that Interaction between UBA and Vpr as well as for the domain in Vpr, which is around 45 Amino acid long UBA domain binds.

1.2.6. Vpr regulates the effects of glucocorticoid hormones

Vpr interacts directly with the glucocorticoid receptor as well as various Transcription factors and can, as a co-activator, the effect of Increase glucocorticoids in different cell lines (Kino et al., 1999). It is known that Vpr is a LXXLL Motif contains how this also occurs in nuclear hormone receptors. Steroid hormone receptor antagonists, such as RU 486, can activate the activator function of Cancel Vpr again. It is believed that Vpr acts as a glucocorticoid coactivator contributes to increased glucocorticoid sensitivity and thereby to the pathogenesis of AIDS.

1.2.7. Incorporation of Vpr into virus particles

In addition to Nef and Vif, which are incorporated in a small number of molecules, Vpr is the only accessory HIV protein that selectively acts in significant amounts selectively via direct interaction of Vpr with the p6 ^gag domain of the Gag polyprotein precursor Pr55 and independently of other viral factors in the Process of virus assembly is incorporated into maturing virions (Cohen et al., 1990; Paxton et al., 1993; Lavallee et al., 1994; Kondo et al., 1995; Lu et al., 1995; Kondo and Göttlinger, 1996) . The areas of Vpr that are required for virus incorporation have been controversially defined: both the N-terminal region (Yao et al., 1995; Mahalingam et al .; 1995, Lavallee et al., 1994; Kondo et al., 1995; Lu et al., 1995) and C-terminal regions (Paxton et al., 1993) were held responsible for this. Positions 36-42 are required for this function within p6gag. Another school, on the other hand, believes that Vpr virus incorporation occurs through interaction of Vpr with the NCp7 domain within the Pr55 gag precursor, an activity that requires the zinc finger region in NCp7 and the C-terminal 16 amino acids in Vpr (Rocquigny et al., 1997).

1.3. Biochemical and structural characterization of Vpr

The only biochemical activity of Vpr characterized in vitro so far is that of a cation selective ion channel (Piller et al., 1996). This work is based on the assumption that the C- terminal helix (positions 46 to 71), which are similar to the bee venom component melittin owns, forms a membrane pore as a transmembrane anchor. In fact, recombinant, in E. coli-expressed Vpr can be reconstituted in artificial planar lipid bilayers. This made one ion channel activity which can be regulated by the membrane potential, the controllability of which is determined by the basic C-terminal region, which corresponds to the negatively charged cytoplasmic side of the Cell membrane should interact.

There is evidence of homooligomerization of Vpr: a recombinant Vpr fusion protein forms oligomeric structures with molecular weights of <100 kDa (Zhao et al., 1994) Observation that has so far not been confirmed on viral Vpr.

To date, studies on the molecular structure of Vpr have been carried out by two groups using secondary structure analyzes on short Vpr peptides: NMR studies on overlapping peptides in aqueous trifluoroethanol (TFE) and alpha-helical regions identified in sodium dodecyl sulfate (SDS) micelles in the Vpr - Positions 50-82. (Yao et al., 1998). The potential for helix formation in the C-terminal as well as the N-terminal region of Vpr has previously been predicted by various authors (Mahalingam et al., 1995a, b; Yao et al., 1995; Wang et al. 1996). Recent studies using CD spectroscopy in TFE-containing solutions on 25 amino acid long peptides (Luo et al., 1998) showed the first experimental evidence for the existence of the N- and C-terminal helices in Vpr ( Fig. 1). The solution structure of a 45 amino acid long peptide Vpr52-96 was recently determined in 30% TFE using 1H NMR, an amphipathic helix was defined in the region 53 to 78 (Schüler et al., 1999).

Numerous and partly controversial mutation analyzes have attempted to assign the different primary and secondary structures to individual biological activities of Vpr (Mahalingam et al., 1995a-d, 1997; Wang et al., 1996; Nie et al., 1998; Di Marzio et al., 1995). The previous model of the structural and functional domains of Vpr is summarized in FIG. 1.

1.4. Chemical synthesis of Vpr peptides and their biological effects

Rocquigny and co-workers first reported the full chemical synthesis of a Vpr protein in 1997. The authors described the synthesis of a 96 amino acid long peptide derived from the virus isolate HIV _89.6 (Collman et al., 1992).

No information is given on the purity or the physicochemical properties of the Vpr peptide. It is shown only by the Far Western blot technique that SDS denatured Vpr peptide interacts with the viral nucleoprotein NCp7 of the same HIV isolate, a finding that has not been observed by any of the numerous other groups working in the Vpr field . A major disadvantage of this Vpr synthesis is the fact that none of the biological activities described so far has been shown by the authors for this peptide. In particular, it is described that this Vpr peptide does not bind to p6 ^Gag , a widely accepted property of Vpr (Paxton et al., 1993; Lavallee et al., 1994; Kondo et al., 1995; Lu et al., 1995; Kondo and Göttlinger, 1996). In addition, it is described that this peptide does not form oligomers, and there are indications that this peptide is insoluble in a purely aqueous system. Likewise, no information is provided on the yield and the synthetic capacity of such a product. In a further study (Roques et al., 1997), the same laboratory presented a model of the Vpr-NCp7 interaction, which is based on structural analyzes of partial sequences of these peptides. However, the data on this are not described in detail in this work.

Partial sequences of Vpr (positions 50-75, 50-82 and 59-86) were used for NMR studies on synthetic peptides (Yao et al., 1998). Another group examined two 25 amino acid peptides from the areas of the predicted alpha-helical domains in Vpr using CD spectroscopy (Luo et al., 1998). Short, about 20 amino acid residues long peptides of the C-terminal region of Vpr, which contain the motif "H ^F / _S RIG", have a concentration of 0.7 to 3 mM cytotoxic effect on various yeast strains such as, for example, Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe (Macreadie et al., 1995, 1996, 1997). Increased concentrations of divalent cations, especially magnesium and calcium, prevent the uptake of the Vpr peptides and thereby their toxic effect. Further studies showed that a C-terminal Vpr peptide (positions 71-82) causes permeabilization, a reduction in the mitochondrial membrane potential and ultimately cell death of CD4 ⁺ T cells (Macreadie et al., 1997). Finally, similar toxic effects have also been demonstrated for total Vpr (Arunagiri et al., 1997). The same recombinant GST-Vpr fusion protein was used for this, which was previously used for ion channel studies on Vpr (Piller et al., 1996). However, the authors report problems with the solubility of the recombinant product in aqueous systems.

1.5. Expression and activities of recombinant Vpr 1.5.1. Expression of Vpr in insect cells

Recombinant Vpr of isolate HIV-1 _NL4-3 was expressed in insect cells after infection with recombinant baculoviruses (Levy et al., 1995). The purification of the product was carried out only by immunoaffinity chromatography on immobilized polyclonal antiserum which was directed against the N-terminal domain of Vpr. Cell culture supernatants were used for this, since recombinant Vpr is secreted non-specifically into the culture medium. Purification strategies for the production of larger amounts of recombinant Vpr have not been described. In most cases, the authors used cell culture supernatants containing Vpr for biological tests. It was shown that recombinant Vpr activates virus replication in PBMC and various latently infected monocyte and T cell lines. The main disadvantages of this procedure are:

• - low yield and no possibility of producing mg quantities of highly pure product;
• - Recombinant Vpr was mixed with detergents in the process of affinity purification, whereby Dialysis and renaturation became necessary;
• - Studies on a possible post-translational modification of Vpr in insect cells have not been carried out described;
• - the effect of recombinant Vpr in HIV-infected primary monocytes / macrophages was not tested.

1.5.2. Expression of Vpr in Escherichia (E.) coli

Expression, purification and biochemical characterization of recombinant Vpr were first described in 1994 by Zhao and co-workers. For this purpose, the coding sequence of the Vpr protein of the isolate HIV-1 _{89.6 was expressed} in E. coli as a fusion protein. For the purpose of purification and detection, a 25 amino acid sequence of the heterologous FLAG epitope was fused C-terminally in this method. Apart from the oligomerization, no information was given on the biological activities of the recombinant product in this work. A major disadvantage of this method is the fact that Vpr is not expressed in its authentic sequence, but as a fusion protein.

In a further method, Vpr of the isolate HIV-1 _{HXB-2 was expressed} in E. coli as a glutathione S-transferase (GST) fusion protein (Piller et al., 1996). After affinity chromatography on glutathione agarose, Vpr was freed of the fusion portion by thrombin cleavage. A major disadvantage of this method is the fact that Vpr has a strong tendency to aggregate after cleavage and cannot be kept in aqueous solution. For example, Arunagiri and co-workers (1997) report that recombinant Vpr produced with these methods became insoluble after the GST fusion fraction had been split off and could therefore only be tested in aqueous systems by maintaining the heterologous fusion fraction. (Bibliography behind the examples).

1.5.3. Patent literature

In patent application WO 95/26361 (Azad, A.A., Macreadie, I.G., Arunagiri, C., 1995) described biologically active peptide fragments of the Vpr protein of HIV; pharmaceutical Compounds containing these peptides or biologically active analogues thereof; Antagonists of the Vpr peptides and pharmaceutical compounds containing these Vpr antagonists. The chemical synthesis of total Vpr protein plays no role in this.  

Subject of WO 96/07741 (Cohen, E .; Bergeron, D .; Checroune, F .; Yao, X.-J .; Pignac-Kobinger, G., 1996) are chimeric molecules consisting of Vpr of HIV-1 and Vpx of HIV-2, which are specific can be built into HIV-1 / HIV-2 virus particles and there the structural organization and disrupt the functional integrity of virions. However, they are for use in gene therapy for HIV 1 / HIV-2 infections excluded.

WO 96/08970 (Weiner, D.B .; Levy, D.N .; Refaeli, Y., 1996) describes methods for inhibiting the Cell division and lymphocyte activation using Vpr proteins, fragments of Vpr or called gene sequences from Vpr. The chemical synthesis of Vpr proteins does not play any part in this Role.

The use of vpr genes in the screening assay for anti-HIV drugs is described in the U.S. Patents 5721104 and 5639619 for determining HIV-2 in US 5580739, a Vpr- Receptor protein in US 5780238.

State the nature of the invention

The object of the present invention is to produce synthetic (s) Vpr proteins which are biological Efficacy as well as the antigenic and physicochemical properties of sVpr proteins demonstrate and thus the principle solution for their use in various areas of HIV to create related basic research, therapy and serodiagnosis.

The task was first solved by the total synthesis of sVpr1-96 from HIV-1 _NL4-3 . This protein differs in 9 amino acid positions from the protein previously described by Rocquigny and co-workers (1997). There is thus a 10% divergence between those already described (de Rocquigny et al., 1997) and the product obtained with the present invention, which contains the total and partial sequences of the Vpr protein of HIV-1 _NL4-3 (Adachi et al., 1986). The product is new, in addition to the method for its production and its novel, advantageous properties.

The new sVpr proteins affect all or part of the sequences of the regulatory HIV protein Vpr. These products are characterized in that their sequence has not yet been described were manufactured in a new way, have a high degree of purity, in large Scale and can be produced with economically advantageous yields as well End products of the manufacturing process are soluble under physiological conditions and none Subject to protein aggregation.

It has been shown that such sVpr proteins are antigenic, biological and physicochemical Have properties comparable to the viral Vpr expressed in HIV-1 infected cells and / or are identical. In particular, it could be shown that sVpr proteins are virus replication activated by various HIV-1 isolates in primary human cells and that in particular the Activity of endogenous viral Vpr can be complemented.

The sVpr proteins according to the invention have antigenic properties - similar to other viral Vpr -  and can therefore be used for the production of high-purity and epitope-specific antibodies become. This is shown by immunization studies using sVpr proteins and later Characterization of the antibodies obtained in this way.

Surprisingly, it was found that the sVpr proteins according to the invention have a assume natural protein folding, are soluble in physiological aqueous systems and thereby for the elucidation of the atomic and 3D structure of Vpr using spectroscopic techniques such as CD (Circular dichroism) and NMR (nuclear magnetic resonance) analyzes as well as using RKSA (X-ray crystal structure analysis) investigations are accessible.

The principle solution of the task is demonstrated using the example of the peptide sVpr1-96 and partial sequences thereof. The peptide sVpr1-96 comprises the authentic amino acid sequence of the native Vpr protein, which is encoded by the vpr gene of the molecular isolate HIV-1 _NL4-3 . Such synthetic Vpr products have not previously been described.

To achieve the object, it is shown within the scope of the invention that sVpr1-96 can activate the virus replication of various T-cell and macrophage-tropic HIV-1 viruses in cultured human blood lymphocytes and thereby in particular in human monocytes / macrophages the loss of viral Vpr in vpr-deficient HIV-1 mutants in trans can complement. Such activity was demonstrated only for ex vivo Vpr and only in HIV-1 infected permanent T cell lines or HIV-1 infected cultures of primary lymphocytes. The activating effect of Vpr has not yet been demonstrated in isolated monocytes / macrophages. An activating effect of sVpr proteins in general and specifically not for sVpr proteins of the isolate HIV-1 _{NL4-3 has also not yet been} reported. As part of the solution to the problem, it is demonstrated for the first time using the example of sVpr1-96 that sVpr proteins have the same or at least similar properties as viral Vpr, are in any case biologically active in cultivated primary cells and are therefore shown for all others in the description of the invention Possible uses of sVpr proteins are relevant.

The object is further achieved by demonstrating that sVpr1-96 has excellent antigenic properties and is therefore suitable as an antigen for immunization for the purpose of producing high-affinity and specific antibodies against various epitopes of Vpr. As a result, sVpr1-96 and partial sequences thereof are suitable for the development of a Vpr antigen (Ag) ELISA (enzyme linked immunoasorbent assay). The N-terminal fragment sVpr1-47 and the C-terminal fragment sVpr48-96 serve as a specific antigen for the production of epitope-different antibodies, which can serve as capture or detection antibodies in the Vpr-Ag ELISA. The total peptide, sVpr1-96, is used to standardize the Vpr-Ag ELISA. Such a serological test system can be used, for example, to determine the concentration of viral Vpr in the blood of HIV-1-infected individuals and thus has potential applications as a prognostic marker for the diagnosis of disease development and the spread of infection in vivo, especially in the therapy and care of AIDS patients . A further solution to the problem is achieved by using sVpr1-96 and the partial sequences in the elucidation of the molecular structure of Vpr. Such studies for total Vpr proteins have not yet been described and, due to the lack of available amounts of highly pure and natively folded Vpr proteins, seem to be hardly possible by the methods described so far. The results of these structural analyzes on sVpr proteins are novel methods which

• a) on the one hand the high concentrations of folded sVpr- necessary for NMR and crystallization Allow proteins to the exclusion of peptide aggregation and on the other hand
• b) through targeted mutations the tendency of Vpr to cis / trans isomeric conformations and suppress the resulting high flexibility of the N-terminal domain of Vpr. Will continue through the use of Vpr binding partners such as
• c) the HIV protein p6 ^gag , which enters into a stable interaction with Vpr in the process of virus assembly or
• d) artificial Vpr binding partners, such as Fab fragments of anti-Vpr antibodies, which stabilize the structure of Vpr in a "natural" conformation. Such stabilization will significantly facilitate the structural analysis of individual domains or of total Vpr and thereby ultimately enable the elucidation of the high-resolution 3D structure of this HIV protein.

So far, it has been described that soluble extracellular Vpr in a relatively low concentration of 0.1 to 10 nM increases the stimulation of virus replication of T-cell tropics and macrophage-tropic HIV-1 viruses in cultures of primary human lymphocytes and CD4 ⁺ T cell lines . Vpr has also been reported to affect the cell division rate of HIV-1 infected and uninfected cells. These phenomena have been described for Vpr proteins, which were obtained either by the expression of recombinant Vpr in baculovirus-infected insect cells or by the enrichment of viral Vpr from peripheral blood of HIV-1 infected subjects (ex vivo). The disadvantage of these studies is therefore that the corresponding Vpr proteins were extracted from different protein mixtures such as blood serum and cell culture supernatants and therefore no highly purified products were used. Because of these circumstances, it cannot be excluded that the activities described so far for Vpr are partly or wholly due to contamination with cellular and / or viral factors. A further disadvantage is the fact that Vpr obtained in this way has potentially been modified by post-translational protein modifications such as phosphorylation, sialinization, ubiquitination and proteolytic processing and is therefore different from Vpr in HIV-infected cells. Another disadvantage is the multiple reported property, in particular of recombinant Vpr expressed in E. coli, that Vpr could only be expressed as a biologically inert fusion protein due to its strong cytotoxic properties. However, even as a fusion protein, Vpr showed a tendency to associate with cellular membranes; the use of ionic detergents for the purpose of purifying recombinant Vpr was necessary. Furthermore, it was reported that recombinant Vpr showed a strong tendency towards protein aggregation after cleavage of the fusion portion and could therefore not be kept in solution in aqueous buffers.

It should also be pointed out that due to its primary sequence Vpr has a strong dipole character: the N and C termini have mutual net charge. In addition, the polar terms are limited by alpha helices, some of which are amphipathic. In addition, it can be assumed that the middle part of Vpr is not structured and thus gives both domains a high degree of flexibility with one another ( FIG. 1). These physicochemical properties are presumably the cause of Vpr's enormous tendency to enter into intra- and intermolecular interactions, a fact which in turn makes it difficult or even impossible to purify Vpr from protein mixtures.

A major advantage for the analysis of the biological and structural properties of Vpr, especially with the aim of characterizing potential antagonists of the Vpr effect, therefore offers the chemical total synthesis of Vpr. To avoid problems that arise in the Relation to previously published methods for obtaining ex vivo or recombinant Vpr originated, in the present description of the invention fully synthetic Vpr- Proteins (hereinafter referred to as sVpr) for the biological described below Functional tests and structural analyzes are used.

According to the invention, a 96 amino acid long sVpr protein is obtained by solid phase synthesis manufactured, highly purified and checked for molecular integrity (hereinafter referred to as sVpr1-96 designated).

The protein sequence of the product sVpr1-96 was derived from the coding sequence of the vpr gene of the molecular virus isolate HIV-1 _NL4-3 .

In addition to sVpr1-96, shorter fragments thereof are also used in the form of sVpr peptides: This concerns the peptides sVpr1-47, sVpr48-96, sVpr1-20 and sVpr21-40 as well as the mutants sVpr1-20 (Asn ^5,10,14 ) and sVpr21-40 (Asn ³⁴ ).

The products according to the invention have the following advantageous properties:

Both the peptide sVpr1-96 and its fragments are characterized by excellent solubility in polar aqueous solutions. Structural analyzes using ¹ H NMR and CD spectroscopy indicate a structured folding of sVpr1-96 and a "ordered" oligomerization with the formation of di- and / or trimeric structures of the peptide. Protein aggregation, as has been repeatedly reported for recombinant Vpr, did not occur for sVpr1-96 and its fragments, even in high concentrations of mM peptide solution.

Furthermore, the essence of the invention resides in the fact that sVpr1-96 in HIV-1 infected cells has a biological effect, as it has so far for other Vpr proteins (ex vivo and recombinant Protein) has already been described. The effectiveness of sVpr proteins and the associated A variety of possible uses are explained in more detail below.

1. Testing sVpr1-96 in cell culture and setting up a test system for searching for Vpr antagonists

In the context of the invention it is demonstrated that sVpr1-96 in cell cultures of primary human  Lymphocytes, PBMC (peripheral monoculear blood lymphocytes), similar to that already described Effect of native Vpr and also in the same concentration range of about 10 nanom Virus replication of T cell tropics and macrophage tropic viruses increases. Was particularly pronounced the Vpr-mediated increase in virus replication in HIV-1 infected macrophages main target cells for Vpr activity in vivo. These results, summarized in the Embodiments 3 and 4 are convincing evidence of the fact that the chemical Synthesis of sVpr1-96 not the biological activities of the Vpr- produced according to the invention Proteins impaired. It should be noted that this is the first description of a biological Function of fully synthetic Vpr is. It is also shown for the first time in the context of the invention that that isolated Vpr in the form of sVpr1-96 acts as an endogenous Vpr in vpr-deficient HIV-1 Can complement mutants in isolated monocytes / macrophages (see embodiment 4).

Due to the biological activities of sVpr1-96 there are many Possible uses of sVpr proteins. This affects the application of sVpr1-96 for the Development of test systems for the determination of potential Vpr-inhibiting agents. For this becomes an infection model with either permanent monocyte cell lines or primary cells developed. Immediately before or after infection of such in vitro cultures with vpr-deficient or - Defined amounts of sVpr1-96 are added to positive HIV viruses, which were previously compared with potential Vpr antagonists were treated. Suppression of the increase mediated by sVpr1-96 virus replication is a direct indicator of the effectiveness of such Vpr inhibitors. The relative simple and also economically justifiable availability of basically unlimited quantities of sVpr1-96, which are used in the test system even in extremely low nanoM concentrations, enables the construction of extensive screening assays.

2. Use of sVpr1-96 for the production of Vpr-specific antibodies and the Development of diagnostics

An immediate use for sVpr1-96 or the synthetic fragments derived from it results from their immunogenic properties, either for the generation of Vpr-specific mono- and polyclonal antibodies or for the detection of Vpr antibodies in the serum of HIV-1-infected subjects. The latter can only be used as a supplement to the classic HIV antibody detection for use in clinical serodiagnostics to identify HIV infection, since only about 30-50% of all HIV-1 infected people develop serum antibodies against Vpr. In contrast, however, sVpr proteins are of great benefit for establishing a two-sided Ag ELISA to determine the concentration of Vpr in the peripheral blood of HIV-1 infected patients. There are indications in the literature that the concentration of serum Vpr correlates with that of the main HIV antigen p24 ^Gag and the status of disease progression. Such a Vpr-Ag ELISA therefore serves as a sensitive forecast marker, which replaces or supplements other, far more complex and cost-intensive methods. This applies, for example, to the polymerase chain reaction to determine the virus concentration in the blood or the "fluorescent activated cell sorting" (FACS) analysis to determine the number of CD4 ⁺ T cells. A Vpr-Ag ELISA for use as a prognostic marker and as a marker for monitoring the effectiveness of an antiviral therapy means enormous savings in terms of time and money.

For the preparation of such a Vpr-Ag ELISA, first N- and C-terminal fragments of sVpr1-96 in the form of the peptides sVpr1-47 and sVpr48-96 for immunization (for example from Rabbit, sheep, mouse) with the aim of obtaining epitope-specific high-affinity mono- and or polyclonal antibodies used. Such Vpr specific antibodies are used in the case of polyclonal immune sera through purification (IgG enrichment and immunoaffinity purification corresponding immobilized Vpr peptides) freed from non-specific antibodies. Serum vpr out Blood samples from HIV-1 infected individuals are labeled as "capture" antibodies to the solid phase of 96-well Plates of immobilized Vpr antibodies bound. The detection of the bound Vpr antigen is then carried out by means of a detection antibody which is linked to certain enzymes, for example Horseradish peroxidase or alkaline phosphatase is coupled. Based on the intensity of the subsequent enzyme reaction, the amount of bound Vpr antigen can be derived. For the Standardization of the Vpr-Ag ELISA is used for sVpr1-96.

Furthermore, sVpr1-96 and its fragments can be used to prepare and characterize Vpr- specific monoclonal antibodies can be used. For this purpose, Balbc mice are first used sVpr1-96 immunized. The production of Vpr-specific hybridoma cell lines follows described standard methods. The epitope characterization of such monoclonal antibodies is done using overlapping sVpr peptide fragments.

Essential for the feasibility of using sVpr peptides according to the invention with The goal of developing immunological test systems is to demonstrate that synthetic Vpr Peptides in the form of sVpr1-96 have immunogenic properties similar to the native Vpr. This is confirmed according to the invention by injecting sVpr1-96 into rabbits and polyclonal anti-Vpr specific sera can be obtained. These antisera have a high titer of Vpr-specific Antibodies and are suitable for the detection of Vpr both in Western blot and by means of Immunoprecipitation. The detection limit for these methods is between 1 and 10 nanoM Vpr. Furthermore, these antibodies can be used for the in situ detection of Vpr by means of immunohistological Detection techniques are applied.

3. Use of sVpr1-96 to elucidate the molecular structure of Vpr

So far, no information is available on the structure elucidation of complete Vpr molecules become. Only individual C-terminal domains of Vpr in the form of approximately 25 amino acids long synthetic peptides were examined by means of CD and NMR spectroscopic studies and Substructures of which have already been described. The reasons for this are presumably lacking on the one hand Availability of larger amounts of pure Vpr protein and, on the other hand, those within the scope of Invention confirmed the fact that proline residues in the N-terminus of Vpr are a cis / trans isomer  Subject to change of conformation.

However, knowing the structure of Vpr is essential to understand the molecular mechanisms of action of this regulatory protein. Again, this information is the Basis for the targeted development of specific Vpr antagonists, which in turn are novel represent antiviral and HIV therapeutic strategies.

According to the invention, the task of structural analysis of sVpr peptides was achieved in that larger amounts of pure Vpr in the form of sVpr1-96 are used for methods of structure elucidation such as RKSA and spectroscopic methods such as CD and NMR. Furthermore, various ways are described with which the high flexibility of the N-terminal domain of Vpr can be restricted or completely prevented. The following results were achieved:

• a) The peptide sVpr1-96 and its fragments can be used in high, milliM- Concentrations are kept in solution. This is an essential requirement for subsequent ones Structural analysis. There is evidence of orderly folding, possibly oligomer formation.
• b) Furthermore, there were clear indications of cis / trans isomerism in one, several or all of the four Pro residues in the N-terminal domain of sVpr1-96 determined. This is a novel finding which conveys the solution for elucidating the structure of Vpr: Problem following strategy:
• c) Study on overlapping peptides, sVpr1-20 and sVpr21-40. This will make the problematic Proline residues identified with a tendency to cis / trans isomerism.
• d) Proline residues identified in this way are replaced by conservative exchanges, usually asparagine, and analyzed in the form of the peptides sVpr1-20 (Asn ^5,10,14 ) and sVpr21-40 (Asn ³⁵ ). These mutants are also examined in the context of total Vpr, as peptide sVpr1-96, with regard to structure and biological functions.
• e) By using structure-stabilizing solution conditions (salt, pH, detergents to stimulate a membrane-like environment) as well as structure-stabilizing factors, such as natural (UBA2 domain of the DNA repair protein HHR23A, p ^Gag domain of the HIV-1 Gag polyprotein precursor Pr55 ^Gag ) and artificial (Fab fragments of Vpr-specific antibodies) binding partners are said to suppress the flexibility of the N-terminal domain and thereby shift the equilibrium to an ordered structure of sVpr proteins.

Due to the functional, antigenic and structural properties described, for sVpr proteins further possible uses:

4. Use of sVpr proteins in the virological basic research of HIV infections, especially to elucidate the molecular pathomechanisms of Vpr

This affects studies of the interaction of Vpr with cellular proteins such as that Glucocorticoid receptor GR-II or studies of Vpr induced mechanisms of apoptosis, G2 arrest and cell differentiation.  

5. Use of sVpr peptides in animal models

With the availability of larger amounts of biologically active sVpr proteins, this is the first time Possibility to test the effects of Vpr or its potential antagonists in to study extensive animal model studies. This particularly affects infection studies Monkeys, e.g. B. Rhesus monkeys that chimeric with monkey immunodeficiency viruses (SIV) or HIV-SIV (SHIV) Viruses get infected.

6. Use of sVpr peptides in the development of vectors for gene transfer in gene therapy

Most of the previously applied / tested methods of gene transfer for gene therapy purposes are based on the integration of a specific gene into the target cell using retroviral vectors. Unfortunately, only lentiviruses of the HIV type, those that contain Vpr and other components of the pre-integration complex, can "meet" the cells that no longer divide. This affects such important tissues as neuronal cells, monocytes / macrophages and other differentiated cells, which are dependent on the mitosis of the possible target cells when using conventional retroviruses for gene transfer methods. An alternative to this is the in vitro assembly of components of the HIV pre-integration complex, consisting of the matrix protein p17 ^MA viral integrase, the viral genome and Vpr.

7. Development of Vpr analogs / antagonists based on D-amino acids

In addition to the advantages already mentioned above, the chemical synthesis of sVpr proteins has the significant further advantage that peptides can be chemically modified in such a way that either all or only desired biological activities are retained. The aim is to change the metabolism and thus the clearance rate of sVpr proteins in vivo and their peptide-based antagonists in the interest of a longer half-life of these products. Such modifications are only possible through total chemical synthesis, but not through recombinant DNA techniques. A conceivable possibility would be, for example, the development of stable cyclic pseudo-peptides that mimic the part of p6 ^gag that interacts with Vpr. If such peptides were taken up by HIV-infected cells, Vpr would no longer be packaged in released virions and could therefore no longer be effective outside the cell. In such a case, for example, the Vpr-mediated infection of monocytes / macrophages would fail and the spread of the infection in the organism would thereby be restricted or completely absent.

The features of the invention emerge from the elements of the claims and from the description out, both individual features and several advantageous in the form of combinations Represent versions for which protection is requested with this document.  

The invention will be explained in more detail by means of exemplary embodiments, without referring to these examples to be limited.

Embodiments Example 1: Synthesis and Purification of Vpr Peptides Example 1a Synthesis of Vpr peptides - General procedure

The C-terminal Vpr peptides were synthesized on a serine resin from Rapp Polymer Tübingen on an ABI 433A synthesizer (Perkin Elmer).

All N-terminal peptides were on a polystyrene-polyoxyethylene carrier resin (TentaGel R-RAM- Resin from Rapp Polymer) synthesized.

The peptides were constructed using FMOC (fluoromethyloxycarbonyl) strategy using following protective groups: O-t-butyl ester for Glu and Asp, OtBu ether for serine, tyrosine and Threonine, Boc (tert-butoxycarbonyl-) for lysine and tryptophan, Trt (trityl-triphenylmethyl) for Histidine, glutamine and asparagine as well as Pbf (2.2.4.6.7-pentamethyl-dihydrobenzofuran-5-sulfonyl-) for Arginine.

After the synthesis had ended, the protective groups were cleaved off using a Splitting mixture consisting of 95% trifluoroacetic acid, the 3% triisopropylsilane and depending on Peptide 2 to 5% ethanedithiol was added. The resin was separated, the reaction solution concentrated and mixed with heptane. It was concentrated again and the remaining oil with diethyl ether digested. The crude peptide was suctioned off and then lyophilized from 10% acetic acid.

Example 1b Purification of the peptides - general regulation

For the purification, 100 mg of crude peptide were each on a preparative HPLC system (Shimadzu LC-8 Plant) chromatographed. All peptides were on a silica gel column (300 × 400 mm Vydac-RP18- Column, particle size 15-20 µ) using a linear gradient consisting of A = 1% TFA (Trifluoroacetic acid) in water and B = 0.1% TFA in 80% acetonitrile with a flow of 100 ml / min cleaned. The eluates were concentrated and lyophilized.

Example 1c: sVpr1-96

The peptide was constructed on a TentaGel S-AC resin (0.20 mmol / gram) on an ABI 433. At the At the end of the synthesis, the FMOC protective group was split off, the resin in succession  Dimethylformamide and methylene chloride washed and dried. The peptide was then in the cleaved from the resin described above and then cleaned.

Molar mass: 11378 found 11381
sVpr1-96:
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His- Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr- Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile- Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile- Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH

Example 1d: sVpr1-47

Analogous to Examples 1 to 3.

Molar mass: 5728 found 5728.8
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-Gln-His-Ile-Tyr-NH ₂

Example 1e: sVpr48-96

Analogous to Examples 1 to 3.

Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg- Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH

Example 1f: sVpr1-20

Analogous to Examples 1 to 3.

sVpr1-20
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH ₂

Example 1g: sVpr1-20 (Asn ^5,10,14 )

Analogous to Examples 1 to 3.

sVpr1-20 (Asn ^5,10,14 )
H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH ₂

Example 1h: sVpr21-40

Analogous to Examples 1 to 3.

Wild type sequence
sVpr21-40
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-NH ₂

Example 1i: sVpr21-40 (Asn35)

Analogous to Examples 1 to 3.

sVpr21-40 (Asn ³⁵ )
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH ₂

Example 1j: sVpr11-25

Analogous to Examples 1 to 3.

sVpr11-25
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu

Example 1k: sVpr41-55

Analogous to Examples 1 to 3.

sVpr41-55
Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala

Example 11: sVpr46-60

Analogous to Examples 1 to 3.

sVpr46-60
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile

Example 1m: sVpr56-70

Analogous to Examples 1 to 3.

sVpr56-70
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile

Example 1n: sVpr66-80

Analogous to Examples 1 to 3.

sVpr66-180
Gln-Leu-Leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg

Example 1o: sVpr76-96

Analogous to Examples 1 to 3.  

sVpr76-96
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH

Example 2 Production of polyclonal anti-Vpr antibodies by immunization with sVpr1-96

5 mg sVpr1-96 were dissolved in H ₂ O, Freund's adjuvant was added and used for standard immunization of a rabbit. The Imunseren thus obtained, designated "R-96", were pooled and portioned frozen.

A dilution series of 0.1 to 10 ng sVpr1-96 was used to test the detection limit of R-96 each with 200 micro-1 human serum from an HIV-1 seronegative subject and 1 micro-L PBS added and then subjected to immunoprecipitation with R-96. In addition, for one Reaction 5 micro-L R-96 each with 30 micro-L Protein-G Sepharose beads (GammaBind-G-Sepharose beads, Pharmacia LKB Biotechnology, Piscataway, NJ, USA). The lysate was previously with Protein-G Sepharose beads loaded with rabbit pre-immune serum pre-cleared. The Precipitation was carried out according to standard conditions already described (Schubert and Strebel, 1994).

The immunoprecipitates were denatured (10 min, 95 ° C) in SDS sample buffer (2% SDS, 1% β-mercaptoethanol, 1% glycerol, 65 mM Tris hydrochloride (pH 6.8)) and in a 12.5% denaturing SDS Polyacrylamide gel (SDS-PAGE, 12.5% acrylic aide gels, FMC Bioproducts, Rockland, ME) was separated ( FIG. 2B). A dilution series of sVpr1-96 (0.01 to 10 nanoM) was also separated in the same gel ( FIG. 2A) The separated samples were electrotransferred to Immobilon polyvenylidene difluoride (PVDF) membranes (Millipore Corp., Bredford, MA), blocked with 5% BSA in PBS (0.3% Tween 20) and then with a 1: 1000 dilution of R The bound antibodies were detected by means of ¹²⁵ I protein G (0.1 mCi / ml; New England Nuclear, DuPont, Wilmington, DE) and subsequent autoradiography ( FIG. 2). The results of the Western blot study show that even 0.01 ng sVpr1-96 can be detected with R-96 without any doubt of sVpr1-96 by immunoprecipitation is in the range of 1 to 10 nanoM sVpr1-96. In other studies (data not demonstrated) it could be shown that R-96 also reacts with viral Vpr, expressed in HIV-1 infected cells, in Western blot as well as in immunoprecipitation.

Example 3 sVpr1-96 increases virus replication and number of living cells in cultures of human peripheral Blood lymphocytes (PBMC)

For infection experiments, parallel cultures of PBMC, which were isolated from peripheral blood of HIV-1 seronegative healthy volunteers, were incubated with phytohemagglutin (PHA) and interleukin 2 (IL-) for two days in accordance with previously published methods (Schubert et al., 1995). 2) stimulates. The lymphocyte _{culture activated in this way was} infected with the same infectious doses of purified viral stock of the T-cell tropical isolate HIV-1 _NL4-3 . Half of the infected culture was treated with 10 nM sVpr1-96, the other with 10 nM solution of a soluble control peptide, Vpu32-81, which has no biological activity in PBMC and was prepared and purified under the same conditions as sVpr1-96 ( Henklein et al., 1993). The control peptide Vpu32-81 comprises the 50 amino acid cytoplasmic domain of the HIV-1 specific virus protein U (Vpu) (Wray et al., 1996). Incubation with the peptides was maintained throughout the experiment, approximately 80% of the cell culture medium was renewed every two days and fresh peptide solutions were added. To determine the amount of virus particles released, samples of the cell culture supernatants were frozen at -80 ° C. At the end of the infection study, the activity of virus-associated reverse transcriptase (RT) was determined in parallel reactions and plotted against time in the histogram ( FIG. 3A). On the basis of the RT profiles, shown in FIG. 3A, it can be clearly seen that in the presence of sVpr1-96, virus replication was increased by a factor of about 2 in the entire course of the spread of infection. This activation of virus production stimulated by sVpr1-96 reaches the maximum two days after that at the time of maximum virus production (day 7 post infection) and then stops with a constant factor. Dose dependence studies (data not shown) showed that this effect occurs at a maximum at a concentration of approximately 10 nM sVpr1-96.

In parallel to the determination of the RT activities, the number of living cells was determined during the infection experiment by exclusion of tryphan blue at every point in time when the medium was changed and plotted against time ( FIG. 3B). This clearly shows that, compared to the control culture, the number of surviving cells during the spread of infection in the culture treated with sVpr1-96 is increased by a factor of about 1.5. In general, the spread of infection in an HIV-infected cell culture is characterized, and can thus also be seen in the control culture ( FIG. 3B), that when the maximum virus production is reached, the cytopathic effect of the virus comes into play and the number of surviving cells rapidly decreases . This is not the case in the presence of sVpr1-96, the cell count only decreases from day 9 post infection and with lower kinetics than in the control culture. Based on this experiment, however, it cannot yet be determined whether this effect is due to the cytopathic mechanisms such as HIV-1-induced apoptosis and / or cell fusion / syncytia formation being suppressed, or whether sVpr1-96 is only the cell number of uninfected cells in the culture increased. It is striking, however, that this effect was not limited to the replication of wild-type virus and also occurred with comparable intensity in PBMC isolates from different donors. For this purpose, PHA-II2-stimulated PBMCs from another donor with the T-cell tropical wild-type virus HIV-1 _NL4-3 ( FIG. 3C) or with the chimeric macrophage-tropical virus NL4-3 (AD8) ( FIG. 3D) infected and incubated with 10 nM sVpr1-96 or the control peptide Vpu32-81. PBMCs were also infected under the same conditions with the vpu-deficient virus NL (AD8) _{-U DEL1} ( FIG. 3E) and with the vpr-deficient virus NL (AD8) deltaR ( FIG. 3F). It is known that both Vpu and Vpr can stimulate virus replication in PBMC. In both cases, a 3 to 5-fold increase in virus replication was observed in the presence of 10 nM sVpr1-96, similar to the situation with wild-type virus HIV-1 _NL4-3 .

In summary, it can therefore be stated that in primary human T lymphocytes sVpr1-96 the virus replication is activated and at the same time the rate of surviving cells within the infected culture is increased. This effect is independent of the endogenous expression of the accessory HIV proteins Vpu and Vpr.

Example 4 sVpr1-96 complements the replication of vpr-deficient HIV-1 mutants in cultures of primary human monocytes / macrophages

In order to test the effect of sVpr1-96 on virus replication in primary human monocytes / macrophages, "monocyte derived macrophages" (MDM) cells were obtained from blood lymphocytes from three different HIV-seronegative healthy individuals (donors # 1 to 3) and were used to Purpose of differentiation cultivated for 14 days. Parallel cultures from the respective MDM isolates were infected with the same infectious doses of the chimeric macrophage-tropical virus NL (AD8) and its vpr-deficient mutant virus NL (AD8) deltaR. At the start of the infection, parallel cultures were incubated with 10 nM sVpr1-96 and as a control with 10 nM Vpu32-81. Every three days, 90% of the culture medium was renewed and new peptide solutions were added to the cultures in question. Aliquots of the culture supernatants were collected over a period of two months and frozen at -80 ° C. Finally, the amount of virus-associated RT activity was determined and plotted against time. The resulting replication profiles are shown in Fig. 4. While productive infection spread in the MDM culture for wild-type virus NL4-3 (AD8) occurred with maximum virus production around day 24 post infection ( FIG. 4A), the replication and the infection spread was the vpr-deficient mutant NL (AD8 ) deltaR significantly reduced: In the MDM cultures of donors # 1 and # 2 ( FIG. 4B, C), this was only about 15% of the wild-type virus, in the MDM culture of donor # 3 no productive infection was even found NL (AD8) deltaR can be found ( Fig. 4D). The limited replication of the vpr-deficient mutant NL (AD8) deltaR is in agreement with the previously described accessory function of Vpr in macrophages / monocytes.

The continuous addition of 10 nM exogenous sVpr1-96 throughout the course of the infection study had no positive effect on the virus replication of wild-type virus NL4-3 (AD8) and the replication profile was even reduced to ~ 60% ( FIG. 4A). In contrast, sVpr1-96 had a clearly stimulating effect on the replication of the vpr-deficient mutant NL (AD8) -deltaR: The virus production in MDM cultures from three different donors was increased several times by sVpr1-96 and was almost identical with the replication profile of wild-type virus in the presence of sVpr1-96 ( Fig. 4C, D). Even in the MDM culture, in which no infection spread of the vpr-deficient mutant NL (AD8) deltaR was observed, sVpr1-96 was able to completely reactivate the virus replication ( FIG. 4D).

In summary, it can thus be stated that (i) in cultivated human Monocyte / macrophage sVpr1-96 did not have a positive effect on the replication of wild-type virus (with existing endogenous synthesis of viral Vpr), but (ii) exogenous sVpr1-96 complement the accessory effects of endogenous Vpr in vpr deficient virus mutants and thus the replication competence of vpr-deficient viruses at the level of wild-type Virus restored.

Example 5 ¹ H NMR spectroscopy on sVpr1-96

The first NMR spectra were recorded in 2 mM solution (17 mg / mL) of sVpr1-96 50% 50% ( ^W / _V ) TFE. The 1D spectrum (as the abscissa and ordinate in Fig. 5) shows relatively broad bands for this small protein. This indicates that under the chosen solution conditions, all or part of the peptide tends to be associated. The peptide was dissolved in aqueous solution, without salt and buffer. The addition of DTT caused no changes in the spectrum, which rules out disulfide binding. The NMR data reveal no evidence of highly oligomeric protein aggregates as was defined for recombinant Vpr with molecular weights of over 100 kDa (Zhao et al., 1994). If such a high molecular form of sVpr1-96 (presumably hexamer) is actually present, then such a molecule would move very slowly and therefore tend towards broad signals. Such a phenomenon was not visible in the spectra of sVpr1-96. Individual spin systems (e.g. 3 × alanine, 1 × valine) could be clearly assigned. Since these spin systems extend over the entire molecule, this is an argument against the existence of hexameric structures. The previous data rather suggest that sVpr1-96 in aqueous solution is mainly present as a balance between mono- and dimeric structures, which can contribute to the sometimes broad signals.

In order to clarify the problem further, 2D TOCSY and NOESY spectra of sVpr1-96 were recorded, which gave indications of cis / trans isomeric forms: Certain areas of sVpr1-96 showed particularly broad signals, while others clearly sharp cross peaks in the 2D Show spectra. In the lower region of the TOCSY spectrum (10-9.3 ppm) three signals from three tryptophan side chains can be seen ( FIG. 5A). Further expansion of these cross peaks allows conclusions to be drawn about heterogeneity; the main signal shows at least two smaller signals. The same observation occurred with histidine ( FIG. 6B) and arginine ( FIG. 6C). Since these three types of amino acid chains occur over the entire length of sVpr1-96 (tryptophan in positions 18, 38 and 54; histidine in positions 33, 40, 45, 71 and 78 and arginine in positions 12, 32, 36 , 62, 73, 77, 80, 87, 88 and 90) and these cross peaks appear as such sharp signals, it can be assumed that a phenomenon other than the previously assumed oligomerization of Vpr (Zhao et al., 1994) for the relatively broad lines in the 1D spectrum are responsible. The individual areas shown in FIG. 6 indicate approximately 20% heterogeneity of the examined signals, which are typical for cis / trans isomerism and are presumably triggered by the four proline residues in sVpr1-96. Such isomeric forms should have a relatively high cis content (up to 40%) for proline residues in positions 14 and 35, since these are close to aromatic amino acid residues.

In order to further investigate the existence of such cis / trans isomers to be defined for the first time in sVpr1-96, the following short peptides are analyzed according to the invention:
sVpr1-20 (1-MEQAPEDQGP ¹⁰ QREPYNEWTL-20) and
sVpr21-40 (21-ELLEELKSEA ³⁰ VRHFPRIWLH-40)

In the following 1D and 2D NMR spectra, multiple signals appeared for different proline residues, which are present in both peptides, sVpr1-20 and sVpr21-40. For sVpr21-40 about 10% cis-protein conformation determined. Similarly for sVpr1-20 signals for three cis-Proline- Structures determined, which together take up about 30% of the total intensity. Summarized it is thus established that cis / trans isomeric forms of proline are the cause of heterogeneous Conformations in sVpr1-96 are. It can be based on the NMR calculations on the short peptides the entire molecule, sVpr1-96, is predicted to be about 40% single cis, 6% double cis and Containing 0.4% triple-cis conformation of all proline residues, only 59% should be in the trans structure available.

In a further step of the analysis, the proline residues are identified, which are mainly related to contribute to this heterogeneity. These proline residues are then shaped by asparagine in sVpr1-96 replaced by a conservative exchange. The goal of this research is to use proline by a similar method To replace side chain position, which has comparable influences on the folding of the Has protein backbones, but is not subject to cis / trans isomerism like proline. The chosen exchange of proline with asparagine is based on previously published studies on the correlation matrix of Amino acid side chains and protein folding (Livingston and Barton, 1996). Such Vpr mutants, which are then no longer subject to cis / trans isomerism are used for structure elucidation by means of NMR and also used for crystallization. The biological activities of such proline-asparagine mutants will be determined later.

Example 6: Material and methods Example 6a: HIV-1 molecular clones and plasmid construction

This has already been published for the production of T cell tropic viruses of the molecular clone NL4-3 Plasmid pNL4-3 (Adachi et al., 1986) used and for the preparation of macrophage tropics Viruses the previously published plasmid pNL4-3 (AD8) (Schubert et al., 95; Freed et al., 1995; Freed and Martin, 1994). This plasmid encodes the molecular clone of a chimeric macrophage tropic  Virus in which the env gene of the primary macrophage-tropical virus AD8 (Theodore et al., 1995) with which the T cell tropic clone NL4-3 was exchanged (Freed and Martin, 1994). For the Replication competence of HIV-1 in monocytes / macrophages is a section of the env gene including the V3 loop, necessary (Schubert et al., 95; Freed et al., 1995; Freed and Martin, 1994; O'Brien et al., 1990; Shioda et al., 1991). To produce the vpr-deficient mutant pNL (AD8) -DR, the plasmid pNL4-3 (AD8) was linearized with the enzyme EcoRI (base pair position 5743), the overhanging ends were filled with DNA polymerase I and then religated. This results in a frameshift mutation in the vpr reading frame, resulting in the expression of one N-terminal fragment of Vpr, which does not have any of the biological activities described so far from Vpr can exercise.

Example 6b: Cell culture

Hela cells were grown in Dulbeccos' modified Eagle's Medium (DMEM) with 10% fetal Calf serum cultivated. PBMCs ("peripheral blood monocuclear cells") were performed by Gradient isolation of blood lymphocytes obtained from healthy HIV-1 seronegative individuals, portioned and frozen in liquid nitrogen until use. Two days before infection PBMC cultures were incubated with phytohaemagglutinin (PHA, 1 mg / ml) and human Interleukin-2 (hIL-2, 20 U / ml) stimulated, the treatment with hIL-2 was then for the continued throughout the cultivation period.

MDM ("monocyte-derived macrophages") were obtained by elutriation in accordance with previously published methods (Schubert et al., 1995; Ehrenreich et al., 1993). Adherent cultures of MDM were first precultivated in DMEM supplemented with glucose (4.5 g / L), penicillin (50 U / ml), streptomycin (50 mg / ml) and L-glutamine (2 mM), sodium pyruvate (1 mM) and 10% human serum according to a modified method by Lazdins et al. (1990). After a 2-week differentiation phase, MDM were resuspended, harvested, replated at a concentration of 0.5 × 10 ⁶ cells / ml and incubated for a further two days before infection.

As a control for potential contamination of the MDM cultures with CD4 ⁺ T lymphocytes, a parallel culture of the prepared MDM isolates was also incubated with the T cell tropic virus NL4-3. Infection and replication of the T cell tropical virus could not be found in any of the MDM cultures used, a fact which attests to the absence of CD4 ⁺ T lymphocytes in all MDM preparations.

Example 7: Transfection and extraction of virus stocks

For the preparation of virus preparations, plasmid DNA from molecular HIV-1 DNA was transfected into HeLa cells using the calcium phosphate precipitation method. For this purpose confluent cultures of Hela cells (5 × 10 ⁶ cells) with 25-microg plasmid DNA in calcium phosphate crystals, produced by a method by Graham and von Eb (1973), were incubated and then a glycerol shock according to Gorman et al. (1982). The cell culture supernatants were harvested two days after transfection to obtain concentrated virus preparations. The cells and their constituents were then separated off by centrifugation (1,000 × g, 5 min, 4 ° C.) and filtration (0.45 microm pore size). Virus particles were pelleted by ultra-centrifugation (Beckman SW55 rotor, 1.5 hr, 35,000 rpm, 10 ° C.) and subsequently resuspended in 1 ml DMEM medium. The virus preparations were sterilized by filtration (0.45 microm pore size) and frozen in portions (-80 ° C). Individual virus preparations were determined by determining the RT (reverse transcriptase) activity, using a test already described (Willey et al, 1988), using [ ³² P] -TTP incorporation in an oligo (dT) poly (A) Standardized template.

Example 8: ¹ H NMR on sVpr

1D and 2D ¹ H NMR spectra were recorded on a DMX 600 Bruker NMR spectrometer without spinning at 300 ° K. The spectra were calibrated on the residual proton of the solvent trifluoroethanol at 3.95 ppm. Further details are given in the legend to the figures.

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Legend to the figures Fig. 1 Structural and functional domains in Vpr

The following primary and secondary structural elements in Vpr are assigned to the amino acid sequence of the protein Vpr of HIV-1NL4-3: negatively charged N-terminus (label ( 1 ), positions 1-17); Helix alpha-1 (marker ( 2 ), positions 18-37); an unspecified region (marker ( 3 ), positions 38-51); Helix alpha-2 (marker ( 4 ), positions 51-76); basic C-terminus (marker ( 8 ), positions 77-96). Overlapping, further areas are shown: a region rich in leucine and isoleucine, which is also referred to as leucine zipper-like or also "LR domain" (marker ( 5 ), positions 60-80); a region containing the repeating motif "HF / SRIG" (marker ( 6 ), positions 71-82); the presumed transmembrane anchor of Vpr, which is necessary for the ion channel activity of Vpr (marker ( 7 ), positions 52-79).

Fig. 2 Immunological reactivity of polyclonal antibodies specific for sVpr1-96 im Western blot and immunoprecipitation

Serum from rabbits immunized with sVpr1-96, R-96, was tested in Western blot (A) and immunoprecipitation (B). A dilution series of 0.01 to 10 ng sVpr1-96 was separated in SDS-PAGE (12.5% acrylic aide gel) (A). A similar dilution series of sVpr1-96 was mixed with human serum, and from this mixture the peptide sVpr1-96 was isolated by immunoprecipitation using the serum R-96, and subsequently also separated in SDS-PAGE (B). After electrotransfer to PVDF membranes, sVpr1-96 was detected using R-96 antibodies and subsequent binding of 125I protein G. The autoradiogram of a 2-day exposure is shown in (A) and (B). The positions of standard molecular weight proteins are on the left, and the positions of non-specific reaction with the heavy (hc) and light chain ( 1 c) of the immunoglobulins used for immunoprecipitation are shown on the right.

Fig. 3 sVpr1-96 activates virus replication and increases the number of living cells in cultures of human PBMC

Cultures of PHA and IL-2 activated PBMCs were followed with equal infectious doses Virus stocks infected: HIV-1NL4-3 (A, B, C), NL4-3 (AD8) (D) and the vpu-deficient mutant NL (AD8) -UDEL1 (E) and the vpr-deficient mutant NL (AD8) deltaR (F). During the Infection experiments, the cultures were in the presence of 10 nM sVpr1-96 or 10 nM des Control peptides Vpu32-81 cultured. The virus release is a profile of the virus-associated RT- Activity shown in the cell culture supernatant (A, C, D, E, F). (B) shows the number of living cells in the Experiment from (A).

Fig. 4 sVpr1-96 activates the replication competence of vpr-deficient HIV-1 mutants in Cultures of primary human monocytes / macrophages isolated from different donors

Parallel cultures obtained from differentiated MDM isolates, from three different donors, were treated with equal infectious doses of purified viral stocks of the macrophage-tropic virus NL4-3 (AD8) and its vpr-deficient mutant NL (AD8) deltaR infected. Virus production was followed over a period of about two months and as a virus-associated RT activity plotted against time.

Fig. 5 2D 1H TOCSY spectrum

(Mixing time = 110 ms) of a 2 mM solution of sVpr1-96 in 1: 1 (V / V) TFE - d2 / H2 = at 300 ° K. The ordinate and abscissa show the corresponding 1D 1H spectra. Magnifications of regions A, B and C are shown in FIG. 6.

Fig. 6

Enlarged regions of the 2D TOCSY spectra, shown in Figure 5, showing the interactions between protons H-7 and H-2 of tryptophan residues (A); H-2 and H-4 correspond to histidine residues (B), and epsilon-H and alpha-H correspond to arginine residues (C).

Claims (24)

1. Use of synthetic peptides of the regulatory virus protein R (Vpr) human Immunodeficiency viruses (HIV) for therapeutic and / or diagnostic purposes. 2. Use according to claim 1, characterized in that it is

• a) a synthetic (s) Vpr protein (sVpr1-96) with 96 amino acids of the following composition:
  H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His- Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr- Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile- Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile- Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH, or
• b) an N-terminal peptide (sVpr1-47) with 47 amino acids of the following composition:
  H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-Gln-His-Ile-Tyr-NH ₂ , or
• c) a C-terminal peptide (sVpr48-96) with 49 amino acids of the following composition:
  Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu- Leu-Phe-Ile-His-Phe- Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH, or
• d) a peptide (sVpr1-20) with 20 amino acids of the following composition:
  H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH ₂ , or
• e) a peptide (sVpr21-40) with 20 amino acids of the following composition:
  H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH ₂ or
• f) a peptide (sVpr1-20 (Asn ^5,10,14 )) with 20 amino acids of the following composition:
  H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH ₂ or
• g) a peptide (sVpr21-40 (Asn ³⁵ )) with 20 amino acids of the following composition:
  H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH ₂ or
• h) a peptide (sVpr11-25) with 15 amino acids of the following composition:
  Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu or
• i) a peptide (sVpr41-55) with 15 amino acids of the following composition:
  Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala or
• j) a peptide (sVpr46-60) with 15 amino acids of the following composition:
  Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile- or
• k) a peptide (sVpr66-80) with 15 amino acids of the following composition:
  Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg or
• l) a peptide (sVpr76-96) with 21 amino acids of the following composition:
  Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH.

3. Use according to claim 1 and 2, characterized in that it is sVpr proteins acts in which the N-terminal domain of the sVpr proteins in one, several or all four Proline residues are mutated. 4. Use according to claim 1 to 3 for the production of poly- and monoclonal Vpr-specific Antibodies or antisera. 5. Use according to claim 1 to 4 for the production of epitope-different Vpr-specific Antibodies. 6. Use of antibodies according to claim 4 and 5 in serological test methods. 7. Use according to claim 4 to 6 in a Vpr antigen (Ag) ELISA. 8. Use according to claim 1 and 7 for the detection and determination of the concentration of viral Vpr in the blood of HIV-infected individuals. 9. Use of sVpr proteins according to claim 1 to 8 as a standard antigen for the calibration of  Vpr-Ag ELISA techniques. 10. Use of sVpr proteins according to claim 1 to 3 for in vitro test systems for determination by Vpr antagonists. 11. Use according to claim 10 for complementing the function of endogenous, viral Vpr in Cell cultures infected with vpr-deficient HIV mutants. 12. Use according to claim 10 and 11 for complementing the function of viral Vpr in Cultures of primary human lymphocytes infected with vpr-deficient HIV mutants are. 13. Use according to claim 10 and 11 for complementing the function of viral Vpr in Cultures of differentiated primary human monocytes / macrophages, which with vpr- deficient HIV mutants are infected. 14. Use according to claim 10 to 13 for the determination of reagents

• a) inhibit the interaction of Vpr with cellular factors such as the glucocorticoid receptor, transcription factors and other DNA-interacting enzymes and factors;
• b) prevent the transcription activating effect of Vpr;
• c) regulate, influence or prevent the activity of Vpr on the action of steroid hormones;
• d) regulate, influence or prevent the transport of Vpr alone or in combination with other components of the HIV pre-integration complex;
• e) regulate, influence or prevent the incorporation of Vpr into virus particles during the assembly of HIV;
• f) regulate, influence or prevent the Vpr-induced cell cycle arrest
• g) regulate, influence or prevent the effect of Vpr on cell differentiation and cell growth
• h) regulate, influence or prevent the cytotoxic effects of Vpr
• i) regulate, influence or prevent the ion channel activity of Vpr

15. Use of sVpr proteins according to claim 1 to 3 for in vivo test systems for determination by Vpr antagonists. 16. Use of sVpr proteins according to claim 1, 2, 3 and 15 in animal model studies for  Determination of functions according to claim 14. 17. Use of sVpr proteins according to claims 1 to 3 for the production of concentrated peptide Solutions. 18. Use of sVpr proteins according to claim 1, 2, 3 and 17 for the production of specific Vpr Antagonists. 19. Use of sVpr proteins according to claim 1, 2, 3, 17 and 18 for reducing the N- terminal domain of Vpr induced flexibility of sVpr by means of structure-stabilizing factors. 20. Use according to claim 19, characterized in that it is the structure-stabilizing factors

• a) the UBA2 domain of the DNA repair protein HHR23A, which binds to Vpr,
• b) Fab fragments of Vpr-specific immunoglobulins or
• c) viral factors, in particular components of the HIV-1 Gag polyprotein precursor Pr55 ^Gag , which come into contact with Vpr, the human glucocorticoid receptor or components thereof, in the process of virus assembly.

21. Use of sVpr proteins according to claim 1 to 3 for in vitro assembly of retroviral Pre-integration complexes. 22. Use of sVpr proteins according to claim 1, 2, 3 and 21 in administrable in vitro or in vivo Gene transfer methods. 23. Use of sVpr proteins according to claim 1, 2, 3, 21 and 22 for transfections, integration in chromosomal and episomal host DNA or other gene transfer methods in eukaryotic Cells. 24. Use of sVpr proteins according to claim 1, 2, 3, 21 and 22 for gene transfers from in vitro manufactured and / or manipulated gene fragments in cells, tissues or organisms with the Purpose of a gene therapy application.