Identifikation und Charakterisierung zellulärer Zielproteine zur antiviralen Therapie der SARS-Coronavirus Infektion

The severe acute respiratory syndrome (SARS) was first observed in
the Chinese province Guangdong in November 2002. The disease
quickly spread around the globe via air travelling and caused a
worldwide epidemic. Several research institutions together with the
World Health Organisation (WHO) identified the SARS-coronavirus
(SARS-CoV) as the causative agent of this disease. During the
epidemic, about 8,000 people were infected with a mortality of
approximately 10%. Although no new infections have been observed
since the summer of 2003, a recurrence of the pathogen cannot be
excluded. Up to now, no specific therapy against the virus have
been available. Viruses contain a very compact genome, which does
not encode all proteins necessary for independant replication.
Thus, viruses necessarily depend on host proteins and have to
interact directly with them. The analysis of protein-protein
interactions between SARS-CoV and human host cells contributes to a
better understanding of the viral replication and pathogenicity.
Prior to this work, an automated, genome-wide yeast-two-hybrid
(Y2H) screen between all 28 proteins of SARS-CoV and the gene
products of three human cDNA libraries had been performed, and
approximately 460, mostly new protein-protein interactions had been
identified. The aim of this work was to confirm newly identified
virus-host SARS-CoV protein interactions and to functionally
analyse them to identify new targets for antiviral therapy. 89
newly identified protein-protein interactions were examined via a
modified LUMIER binding-assay to confirm individual interactions.
37 out of 89 protein interactions were found to be positive,
resulting in a confirmation rate of 42%. In subsequent functional
analyses of protein-protein interactions between the SARS-CoV
non-structural protein 1 (Nsp1) and proteins of the immunophilin
family, two different functional consequences were observed. First,
it could be shown that SARS-CoV Nsp1 boosts the expression of genes
regulated via the calcineurin/NFAT-signalling cascade. The
increased expression of NFAT-regulated genes in SARS-CoV infection
may cause the cytokine dysregulation described in SARS patients
which leads to severe lung tissue destructions and which correlates
with high mortality. The considerably less harmful human
coronavirus HCoV-NL63 and mouse coronavirus (MHV) did not boost the
expression of NFAT-regulated genes. It was thus hypothesized that
the therapy of the cytokine dysregulation with the
immunosuppressive drug Cyclosporine A (CspA) might improve the
course of the disease. In addition, it could be shown for the first
time that the replication of the SARS-CoV can be inhibited by the
immunosuppressive drug CspA. Subsequent experiments showed a
similar inhibition of the viral replication of the less harmful
human coronavirus HCoV-NL63 and HCoV-229E mediated by CspA. In
cooperation with several groups of the ”SARS-Zoonose- Verbund”,
further inhibition experiments were performed with animal
coronaviruses like FCoV, IBV Bd and TGEV PUR46, which showed a
similar antiviral effect of CspA. The two cellular proteins
Cyclophilin A and FK506 binding-protein 1A were shown to be
essential for viral replication of HCoV-NL63. The findings of this
work may contribute to a better understanding of the interactions
between SARS-CoV and infected host cells and their innate immune
response. The application of the general coronaviral inhibitor CspA
identified in this study and of non-immunosuppressive CspA
analogues like DEBIO-025 procures promising options for
anti-coronaviral therapy.