European Journal of Medical Research - Deutsche AIDS ...

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.