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Research Group PD Dr. rer. nat. Dr. habil. med. Albrecht von Brunn

From intra-viral and virus-host protein-protein interactions to broad-spectrum antivirals (of Coronaviruses)

Areas of investigation

Coronaviruses (CoVs, family Coronaviridae, subfamily Coronavirinae) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. Six distinct CoVs (HCoV-NL63, HCoV-HKU-1, HCoV-OC43, HCoV-229E, SARS-CoV, HCoV-MERS) cause respiratory tract illness in humans, ranging from mild common cold infections in immune-competent individuals to deadly virus-associated pneumonia and kidney failure. At least seven different animal CoVs cause economically significant epizootics in livestock, and deadly disease in companion animals. Some CoV have zoonotic potential and can be considered as emerging viruses. The recent and ongoing outbreaks of severe acute respiratory infection caused by the emerging SARS- and MERS-CoVs, respectively, demonstrate the necessity to develop highly effective, broadly-acting drugs against zoonotic viruses.
My laboratory studies the interplay between viral and host cell proteins of coronaviruses in order to identify and understand cellular determinants and pathways responsible for the different pathogenicities. The goal is to identify common targets as broad-range antivirals.

1. From genome to orfeome to intraviral and virus-host interactome

CoVs are enveloped viruses carrying the largest known single-stranded RNA genomes (25–32 kb) with positive-sense orientation (Figure 1). The first two thirds of the genomes encode two polyproteins ORF1a/ORF1b, which are processed by viral proteases into 16 non-structural proteins with various enzymatic functions required for genome replication. The last third of the genome contains four structural proteins S, E, M, N and - depending on the virus – various accessory genes. To understand different pathogenicities of the CoV family members it is essential to gain knowledge on the function of the individual viral proteins, their interacCoVs are enveloped viruses carrying the largest known single-stranded RNA genomes (25–32 kb) with positive-sense orientation (Figure 1). The first two thirds of the genomes encode two polyproteins ORF1a/ORF1b, which are processed by viral proteases into 16 non-structural proteins with various enzymatic functions required for genome replication. The last third of the genome contains four structural proteins S, E, M, N and - depending on the virus – various accessory genes. To understand different pathogenicities of the CoV family members it is essential to gain knowledge on the function of the individual viral proteins, their interaction with cellular proteins and the consequences of these interactions on cellular signaling pathways.

Figure 1. Genome organization of SARS-CoV.
Figure 2: Functional categories derived from Y2H data of cellular interaction partners of SARS-CoV proteins and of networks of direct (level 1) cellular interactands (red) and indirect (level 2) interactands (orange) with SARS-CoV proteins (brown).

Similar screenings are being performed with further CoVs (e.g. HCoV-NL63). We anticipate to identify cellular molecules and pathways explaining different pathogenicities of the viruses and serving as common antiviral targets.

Using high-throughput yeast two-hybrid screening technologies, our lab was one of the first to study protein/protein interactions (PPIs) of individual SARS-CoV proteins at intra-viral, matrix-based (screening all viral proteins against each other) as well as virus-host levels (screening viral ORFs against human cDNA libraries; Figure 2). Viral Orfeomes are constructed by PCR amplification of all ORFs and a number of sub-fragments lacking transmembrane regions of the respective CoV from cloned viral DNA. Fragments are cloned into GATEWAY™- compatible pDONR vectors. From here they can easily be shuttled into pro- and/or eukaryotic (Y2H, mammalian) destination expression vectors. Mammalian gene products interacting with viral proteins are identified by screening individual CoV ORFs against human, yeast-expressed cDNA libraries. Positive hits are confirmed in mammalian cells by a variety of techniques including split YFP, LUMIER-, CoIP assays, and in collaboration with several labs Bimolecular Fluorescence Complementation, split-YFP, Fluorescent Three Hybrid, Mass Spec, X-ray crystallography. We have available a battery of expression vectors with various N- and C- terminal fusion tags (e.g. HA, c-myc, HIS, GFP, RFP, split YFP).

2. Involvement of immunophilins in CoV replication

Figure 3: Plaque Titration of a CoV (here SARS-CoV) in the presence of various concentrations of cyclosporin A. The virus causes plaques in a monolayer of CaCo2 cells indicating the number of infectious virus particles. At increasing concentrations of CsA virus particles decrease. They are not detectable any more at optimal inhibitor concentration.

The power of our Y2H screening approaches for successful signaling pathway and antiviral target identification is illustrated by the identification of immunophilins (cyclophilins and FK506-binding proteins [FKBPs]) as interaction partners of non-structural protein Nsp1. These molecules modulate the calcineurin A (CnA)/NFAT (Nuclear Factor of Activated T cells) pathway, which plays an important role in immune-cell activation. They further display peptidyl-prolyl isomerase (PPIase) and chaperon activities helping cellular and some viral proteins to be correctly folded.

Possible roles of Cyclophilins/FKBPs and the CnA/NFAT pathway in the context of CoV replication
We identified Nsp1 of human CoVs to bind to cellular cyclophilins and FKBPs. This has consequences on the important immunological CnA/NFAT signaling pathway: CnA and NFAT represent key molecules for regulation of immune genes, especially interleukins. CnA is activated by calcium-calmodulin-dependent signaling and dephosphorylates NFAT in activated T-cells. De-phosphorylation is a prerequisite for the translocation of NFAT into the nucleus, where it exerts transcriptional influences on NFAT regulatory sequences. As a coincidence of nature, this cascade can be blocked by Cyclosporin A (CsA) and FK506 (Tacrolimus), which bind in complex with cyclophilins and FKBPs, respectively, to CnA. These complexes block CNA phosphatase activity on NFAT. Consequence is the block of the cellular branch of the immune system, i.e. immunosuppression. Overexpression of Nsp1 and infection with SARS-CoV strongly increase signaling through the CnA/NFAT pathway and enhance the induction of IL-2, compatible with late-stage immuno-pathogenicity and long-term cytokine dysregulation observed in severe SARS cases.

Possible roles of Cyclophilins/FKBPs as broad-spectrum inhibitors of CoV replication
Cyclophilins and FKBPs represent large families of peptidyl-prolyl cis/trans isomerases (PPIases) with chaperone-like activities thus excerting important functions on folding, maturation and trafficking of proteins within the eukaryotic cell. CsA acts as a tight-binding, reversible, competitive inhibitor of the PPIase activity. Cyclophilins directly interact with cellular proteins, and in some cases also with viral proteins thus granting replication sensitivity to CsA (e.g. HIV, HCV).
For CoVs we have found that inhibition of cyclophilins by CsA block the replication of representatives of all genera, including human SARS-CoV (Figure 3), HCoV-229E and -NL63, feline CoV, Mouse Hepatitis Virus (MHV) as well as avian infectious bronchitis virus. For HCoV-NL63, we showed that the virus is not able to grow in a CaCo2 Cyclophilin A (CypA)-knockdown cell line, an observation indicating a crucial role of this cellular protein in virus replication. HCoV-229E does not or very efficiently replicate in human hepatoma Huh7.5 CypA knockdown cells or in the same cells reconstituted individually for SNP mutations located in the vicinity of the active enzymatic center of CypA. Similarly, we have shown that replication of human CoVs SARS-CoV, HCoV-NL63, and HCoV-229E is inhibited by the drug FK506 and that the cellular proteins responsible are FKBPs. The results indicate that these Host-Targeting-Agents (HTAs) might serve as broad-range inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

3. Influence of tumor suppressor protein p53 on coronavirus replication

The protein/protein interactions screenings at virus-host level revealed an indirect link between the SARS-CoV SUD and PLPro domains of non-structural protein nsp3 and p53: we found that both CoV domains bind to and stabilize the host E3 ubiquitin ligase RCHY1. This enzymes marks proteins by ubiquitination (addition of small ubiquitin protein molecules) thus changing their properties. One consequence is the degradation of the ubiquitinated proteins in the host-cell proteasome complex. p53 is one of the targets of RCHY1. p53 regulates a number of target genes that mediate tumor suppression. For some viruses it deploys anti-viral activity. We tested a possible functional link between p53 and SARS-CoV replication by infection cells lacking p53. Indeed, we found that in p53-negative cells replication of SARS-CoV was several orders of magnitude more efficient than in p53-expressing cells. As p53 regulates genes involved in the non-specific antiviral defense system (innate immunity) we believe that the degradation of p53 is actively promoted by viral domains stabilizing the RCHY1 enzyme.

4. Future directions

Antiviral drugs are conventionally directed against viral enzymes/proteins. The selective pressure to mutate e.g. active centers of replicative enzymes or proteases is very high. Our philosophy is that this is not the case for cellular protein targets because the respective genes of the infected eukaryotic cell do not "mutate away” easily, thus changing amino acids and protein structures upon selective drug pressure. The barriers for developing host-factor resistance can be expected to be much higher. Therefore, we are following up a number of projects characterizing different protein-protein interactions of viral and cellular counterparts identified by our various screening projects.
Regarding possible applications of immunophilin inhibitors as antivirals, it is not desirable to apply immunosuppressive drugs for the treatment of virus infection. CsA and FK506 molecules can be chemically synthesized and modified such, that they lose their CnA-binding properties in concert with cyclophilins or FKBPs, but not the affinity to the PPIase enzymatic functions. We test such non-immunosuppressive derivatives (e.g. Alisporivir=Debio-025 or NIM811) in cell culture for their inhibitory potential on viral replication. It is highly expected that our results will be translated into the preparedness against epidemic or pandemic emerging viruses.

p53 poses completely new aspects on the first line of defense against CoV infection. We will investigate viral ORFs, cellular genes and mechanisms involved in these processes.

Current group members

Albrecht von Brunn, PD Dr. rer. nat. Dr. habil. med.
Email: vonbrunn(at)mvp.uni-muenchen.de
Phone: +49 89-2180-72839

Dev Raj Bairad, predoctoral student
Email: Bairad(at)mvp.uni-muenchen.de
Phone: +49 89-2180-72872

Brigitte von Brunn, MTA
Email: vonbrunn_b(at)mvp.uni-muenchen.de
Phone: +49 89-2180-72872

Sebastian Schwinghammer, MD student (FöFoLe programme)
Email: schwinghammer@mvp.uni-muenchen.de
Phone: +49 89-2180-72872

Yue Ma-Lauer, Dr. rer. nat., Dipl. Biol.
Email: ma_lauer(at)mvp.uni-muenchen.de
Phone: +49 89-2180-72872/72839

Konstantin Pusl, MD student (FöFoLe programme)
Email: pusl(at)mvp.lmu.de
Phone: +49 89-2180-72872

Former lab members

Carbajo, Javier, TA
Decker, Manuela, B.A.
Huber, Jürgen, Dr. med., Arzt
Knüppel, Larissa, B.A. Biochemistry student
Kurz, Marisa, MA Biochemistry
Mayerhofer, Peter, MA Biochemistry
Mingxia, Su, Dr. rer. nat.
Schöpf, Julia, Dr. rer. nat., Dipl. Biol.
Senninger, Nicole, MTA
Sienel, Wulf, Dr. med., Arzt
Stellberger, Thorsten, Dr. rer. nat., Dipl. Biol.
Teepe, Carola, Dr. med., Ärztin
Wermke, Nadja, TA
Wizemann, Harald, Dr. rer. nat., Dipl. Biol.

Awards and Honours

  • Graduate Student Scholarship, The University of Texas at Austin, Austin, TX, USA, 1981-1982
  • Marine Biological Laboratory, Woods Hole, MA, USA, Walter E. Garrey Scholarship, 1983
  • Nachwuchsförderpreis der Gesellschaft zur Förderung der Molekularbiologie Heidelberg e.V. (GFM), 1989

Contact

PD. Dr. rer. nat. Dr. habil. med. Albrecht von Brunn
Max von Pettenkofer Institute, Virology
National Reference Center for Retroviruses
Faculty of Medicine
LMU München
Pettenkoferstr. 9a
80336 Munich, Germany
Tel.: 089-2180-72839
Fax: 089-2180-72902
vonbrunn(at)mvp.uni-muenchen.de

Invaluable collaboration partners

  • Prof. Christian Drosten, Dr. Marcel Müller, Dr. Doreen Muth, Dr. Susanne Pfefferle, Rheinische Friedrich-Wilhelms-Universität Bonn - Medizinische Fakultät - Institut für Virologie
  • Prof. Rolf Hilgenfeld, Institut für Biochemie, Universität Lübeck
  • Prof. Gunter Fischer, Dr. Miroslav Malešević, Martin-Luther-Universität Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Division of Enzymology
  • Prof. Heiko Hermeking, Institut für Pathologie, LMU München
  • Prof. Heinrich Leonhardt, Department Biologie II, LMU München
  • Prof. Matthias Mann, Dr. Marco Hein, Max-Planck-Institute für Biochemie, Martinsried
  • Prof. Gottfried Pohlentz, Prof. Johannes Müthing, Universität Münster
  • PD Dr. Christel Schwegmann-Weßels, Stiftung Tierärztliche Hochschule Hannover, Institut für Virologie

Former collaborators on projects

  • Prof. Ralph Baric, Rhonda Roberts, University of North Carolina at Chapel Hill, NC, USA
  • Prof. Jürgen Haas, Max-von-Pettenkofer-Institute, LMU München
  • Prof. Georg Herrler, Stiftung Tierärztliche Hochschule Hannover, Institut für Virologie
  • Dr. Manfred Kögl, DKFZ, Heidelberg
  • Prof. Rainer Pepperkok, Dr. Jeremy Simpson, EMBL Heidelberg
  • Prof. Stefan Pöhlmann, Medizinische Hochschule Hannover, Laboratoriumsmedizin/ Virologie
  • Prof. Heinz-Jürgen Thiel, Justus-Liebig-Universität Gießen, Veterinärmedizin, Institut für Virologie
  • Dr. Volker Thiel, Kantonsspital St. Gallen
  • Dr. Peter Uetz, Forschungszentrum Karlsruhe
  • Prof. Friedemann Weber, Albert-Ludwigs-Universität Freiburg, Institut für Medizinische Mikrobiologie und Hygiene
  • Prof. Ralf Zimmer, Prof. Caroline Friedel, Institut für Informatik, LMU München