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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 115 E.3 (Poster) Mechanisms of HIV–1 resistance to a membrane-bound entry inhibitor Hermann F. 1 , Lohrengel S. 2 , Dittmar M. 2 , Dietrich U. 1 , von Laer D. 1 1 Georg-Speyer-Haus, Angewandte Virologie und Gentherapie, Frankfurt, Germany, 2 Universitätsklinikum Heidelberg, Hygiene-Institut, Heidelberg, Germany Objective: HIV entry into CD4 positive T cells is mediated by its trimeric envelope (env) surface protein. Env consists of the receptor binding subunit gp120 and the transmembrane subunit gp41 which facilitates the fusion of viral and cellulare membrane. The process of virus binding and fusion is accompanied by extensive conformational rearrangements within the env protein. The conformational changes in gp41 lead to the formation of the 6-helix bundle, which is crucial for the fusion process. So called C-peptides (e.g. T-20), which are derived from gp41 are efficient inhibitors of viral fusion as they prevent 6-helix bundle formation. In our group a retroviral vector (M87o) has been developed encoding a membraneanchored C-peptide (maC46). maC46 is highly active even against T-20 resistant virus strains. It was the aim of this project to raise a maC46 resistant virus strain. The resistant virus was genetically as well as phenotypically analyzed. Methods: The CCR5-tropic virus strain BaL sel. MD (resistant to a membrane-bound version of T-20) was passaged on different retroviraly transduced PM-1 T cell lines for more than 150 days, which expressed maC46 at increasing levels. The env gene sequence of the passaged virus was determined. The mutant env was used to pseudotype lentiviral vectors. The particles were used for various single round infection assays to analyse the mechanism of resistance. Results: The maC46 resistance of the selected virus was only weak (app. 5-7 times compared to the parental strain). 5 mutations leading to aa changes could be identified within the env gene of the passaged virus. 3 alteration within gp120 (I187; N305, E353) and 2 mutations in gp41 (V556, A579) could be found. The mutations within gp41 were not sufficient to produce the resistant phenotype. The selected virus shows an increased fusion kinetic, although coreceptor affinity is reduced. Conclusion: It has been previously reported the fusion kinetic influences the sensitivity of HIV to C-peptide fusion inhibitors, but this is the first time that it could be shown that HIV actually employs this mechanism to gain resistance. We could show that the some resistance mutation have a global effect and lie far away from the region which is targeted by maC46 within gp41. E.4 (Vortrag) Selection of peptides inhibiting different steps in the HIV-1 replication cycle from phage displayed peptide libraries Dietrich U. 1 , Dietz J. 1 , Hüther A. 1 , Dervillez X. 2 , Kräusslich H.-G. 3 , Königs C. 4 , Humbert M. 5 1 Georg-Speyer-Haus, Frankfurt, Germany, 2 Hôpital Pitié Salpêtrière, Paris, France, 3 University Clinics, Virology, Heidelberg, Germany, 4 JW Goethe University, Pediatrics III, Frankfurt, Germany, 5 Dana Farber Cancer Institute, Boston, United States of America Objectives: The aim of this study was the selection of peptide ligands for different viral and cellular targets in order to interfere with HIV-1 replication at various steps of the replication cycle. Methods: Recombinant target proteins for the selection of peptide ligands were expressed in E. coli or eukaryotic cells. Peptide ligands for various immobilized targets were selected from phage displayed peptide libraries (NEN Biolabs) that express peptides of 7 or 12 amino acids in length in a linear or cyclic form fused to the phage pIII protein. Specificity of binding of the selected phages was confirmed by ELISA and the peptide sequences were deduced from the corresponding nucleotide sequences of the phage genomes. The selected peptides were tested for antiviral activity against HIV-1 in functional cell culture assays (single round infection assays, marker gene transduction assays). Results: We could select peptide ligands for various target structures including HIV-neutralizing antibodies, the viral envelope protein gp120, the highly structured psi-RNA containing the packaging signal for viral genome encapsidation as well as for the multimerizing domain of Gag. Some of the peptide ligands were optimized by mutagenesis or their functionality was improved by multimerization. Functional analyses of the selected peptides revealed interference with HIV-1 replication at different steps of the viral replication cycle (entry, packaging, multimerization) depending on the target structures used for selection. Conclusion: The selected peptides represent good candidates for the derivation of peptidic or non-peptidic antiviral molecules. E.5 (Vortrag) Novel role for HIV-1 transframe protein p6* in viral replication Leiherer A. 1 , Ludwig C. 1 , Wagner R. 1 1 Universität Regensburg, Institut für Medizinsche Mikrobiologie und Hygiene, Molekulare Mikrobiologie und Gentherapie, Regensburg, Germany Objective: The HIV-1 transframe protein p6* is encoded by the very 5´end of the pol gene and so its reading frame overlaps with p1 and p6 in the gag readingframe and the highly conserved ribosomal frameshift site. The p6* protein is located between nucleocapsid (NC) and protease (PR) in the Gagpol polyprotein precursor. Besides its aminotermius, also the carboxyterminal portion of p6* is highly conserved and has been reported to play a role in regulation of HIV-1 protease activation. Previously it has been suggested that the Nef protein, a key player of HIV-1 infectivity and progression to AIDS, binds to p6* to facilitate viral replication. This prompted us to analyze the role of p6* for HIV-1 replication. Methods: A panel of recombinant NL4-3-derived proviruses either containing or lacking nef was generated comprising clustered mutations throughout the p6* coding region without manipulating the gag open reading frame or affecting proper frameshifting or release of the viral protease. Besides, the role of a central p6* domain duplicated in the nef-negative virus HX10 was addressed by inserting this region into wild-type NL4-3. The effect of these mutations on viral infectivity and replication has been examined in cell culture. Results: Partially mutated p6* variants did not significantly influence viral infectivity or replication of the corresponding viruses. However, mutation of the entire p6* region decreased significantly viral infectivity irrespective of nef expression. Whereas this fully mutated p6* delayed the replication of nef-
116 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 containing viruses, replication was completely abolished in established cell lines in the absence of nef. Furthermore, viral infectivity of nef-positive and -negative NL4-3 viruses was enhanced by the HX10-derived duplication of the central p6* domain which appeared to partly rescue compromised infectivity of nef-lacking viruses. Of note, incorporation of Nef in viral particles was unaffected by any mutation in p6*. Conclusion: Our results suggest a new role for p6* in viral replication associated with Nef function and further experiments are underway to clarify the importance of distinct p6* regions for potential Nef interaction. E.6 (Poster) Generation of soluble multimeric peptides for inhibition of HIV-1 entry Hüther A. 1 , Krüger T. 1 , Daelken B. 1 , Wels W. 1 , Königs C. 2 , Dervillez X. 3 , Dietrich U. 1 1 Georg-Speyer-Haus, Institute of Biomedical Research, Virology, Frankfurt am Main, Germany, 2 JW Goethe University, Department of Pediatrics III, Frankfurt am Main, Germany, 3 Hôpital Pitié Salpétrière, Paris, France Background: To improve the antiviral activity and the stability of antiviral peptides, we expressed them as soluble multimers in a eukaryotic or prokaryotic expression system. These systems also allow to potentially combine different antiviral functions and, in the case of expression in eukaryotic cells, to introduce posttranslational modifications. We used this system to express a variety of peptides inhibiting HIV-1 entry. Methods: The eukaryotic expression system was established by expressing the fusion inhibitory T20-related peptide C46 (Dervillez et al. 2006). We then expressed peptides corresponding to each of the extracellular loops of CCR5, peptides mimicking HIV-1 entry domains selected by phage display and the CDR3 region of pE51 mAB binding to the co-receptor binding site of gp120 like CCR5. Constructs were cloned into the pEF-IRES expression vector between a signal peptide and the human C4bp multimerising domain together with His and Myc-tags for purification and detection. For bacterial expression, constructs were cloned into the pFLAG vector and expressed in E.coli Trx BL21. After affinity purification, the antiviral activity of the multimers was analysed in single round HIV-1 entry assays. Results: The different multimeric extracellular CCR5-constructs were expressed, purified and characterized biochemically. All constructs are heptameric like the natural human C4bp protein. Entry-inhibition assays with JR-FL and D117III HIV-1 pseudoviruses and U87-CD4-CCR5 cells showed improved inhibition of HIV-1 entry for most of the multimeric constructs as compared to their monomeric form. This increased potency was very prominent for the multimeric N-terminal CCR5 and the E51 peptides which have an IC50 of 90-120 nM in contrast to the monomers (high micromolar range). Conclusion: The antiviral activity of short peptides can be improved by multimerization. Further improvement of entry inhibition may be possible by combining peptides interfering with different steps of HIV-1 entry. Furthermore, preliminary data also point at improved half-life in vivo and a more favourable tissue distribution and may therefore allow to reduce the number of applications. This work was supported by the EU-6th FWP TRIoH LSHG-CT- 2003-503480. E.7 (Poster) Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation Brandt S. 1 , Blißenbach M. 1 , Grewe B. 1 , Grunwald T. 1 , Überla K. 1 1 Ruhr-Universität Bochum, Molekulare und Medizinische Virologie, Bochum, Germany Objective: The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Methods / results: Using Rev-independent gag-pol expression plasmids of HIV-1 and SIV and lentiviral vector constructs, we have now observed that HIV-1 and SIV Rev enhanced RNA encapsidation 20 to 70-fold correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. Conclusions: In addition to specific interactions of nucleocapsid with the packaging signal at the 5´end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA. E.8 (Poster) Identification of SH3 domain-containing proteins involved in HIV-related signal transduction pathways Asbach B. 1 , Ludwig C. 1 , Wagner R. 1 1 Universität Regensburg, Institut für Medizinsche Mikrobiologie und Hygiene, Molekulare Mikrobiologie und Gentherapie, Regensburg, Germany Background: The long-term efficacy of vaccine-candidates as well as therapeutics targeted against HI-viral proteins suffers from the virus´ high variability. During its life-cycle HIV hijacks various cellular systems and interferes with the equilibrium of the cell´s signalling status in favour of viral fitness, e.g. by downregulation of CD4 expression. Consequently, there are many interactions between viral and cellular proteins against which therapeutic vaccines can be directed, that are focused on cellular side. Objectives: Our goal is to identify new interactions in the cell´s complex signalling network, which are crucial for successful HIV replication, focussing especially on SH3 domaincontaining proteins. The src-associated protein in mitosis of 68 kDa (Sam68), which contains numerous potential target sites for binding of SH3-domains, has been shown to be a necessary host-factor for HIV-replication, playing an important role in the nuclear export of late viral transcripts. Our aim is to identify new SH3 interactors of Sam68 and to determine their importance in modulating the functions of Sam68 in HIV-replication. Methods: We applied a phage display technique to identify SH3-binders of Sam68. As bait we used recombinant Sam68 purified from E.coli. The phage library consisted of all 296 human SH3-domains as fusions with M13-surface-protein pVIII.
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