This project funded by the BayGene program (Bayerisches Genomforschungsnetzwerk) focuses on several different herpesvirus species and aims at the identification of protein interactions that are either conserved between herpesvirus species or virus-specific and can be targeted for therapeutic purposes. In five herpesvirus species (HSV-1, VZV, mCMV, EBV, KSHV) for example, we identified 1,252 intraviral protein interactions and found that interactions between core proteins are more highly conserved than their actual sequence homology suggests (Fig. 5). For two of these herpesviruses, VZV and KSHV, we also identified 1,128 virus-host protein interactions and characterized major principles of the interaction between pathogen and host proteomes (Fig. 6). To generate multi-dimensional, highly comprehensive models of virus-infected cells, the yeast-two-hybrid analyses are complemented by ongoing projects using other genome-scale, high-throughput technologies: (i) genome-scale siRNA knock-down screens in collaboration with Prof. Peter Ghazal, University of Edinburgh, (ii) large-scale structural analyses in collaboration with Prof. Paer Nordlund, Karolinska Institute, (iii) proteome expression and generation of monoclonal antibodies in collaboration with Prof. Stipan Jonjic, University of Rijeka, (iv) small molecule screens for inhibitors of herpesviral protein interactions in collaboration with Prof. Ulrich Koszinowski, Max-von-Pettenkofer Institute (Virology Dept.) and Prof. Garry Taylor, University of St Andrews.

Crucial aspects of the herpesviral replication cycle including DNA replication, gene transcription and RNA export, capsid morphogenesis, and egress occur in the host nucleus and require that numerous herpesviral proteins are transported into this compartment and some of them back to the cytosol. Moreover, a considerable part of viral activity targeted at the cellular machinery takes place in the cell nucleus. In general, transport of proteins between cytoplasm and nucleus is mediated by direct or indirect binding to transport factors of the importin -family and occurs along a gradient of the small GTPase Ran (Fig. 7). So far the mechanism of nucleo-cytoplasmic transport is unknown for a large number of viral proteins. In this project funded by the DFG we will apply novel, high-throughput based technologies to establish a comprehensive map of nucleo-cytoplasmic exchange of all herpesviral proteins by screening the complete set of viral proteins of five different herpesviruses for interaction with all transport factors of the eucaryotic importin -family. The regulated spatio-temporal distribution of viral proteins between nucleus and cytoplasm is vital for viral replication, and analysis of the underlying mechanism(s) is an important step towards a comprehensive model of herpesviral infection.

Herpesviral capsids are generated in the host nucleus and need to travers several membrane compartments to acquire their final envelope. Egress from the nucleus starts with budding of capsids at the inner nuclear membrane (INM) (Fig. 8). Capsids with a primary envelope temporarily remain in the perinuclear space until they are de-enveloped at the outer nuclear membrane and reach the cytoplasm to be equipped with additional tegument proteins and finally enveloped at the trans-Golgi network. Processes associated with both primary and secondary envelopment are most likely complex in nature and recruit numerous viral and cellular proteins: Nuclear egress depends on two conserved and essential proteins of the UL34/UL31 family of herpesviral proteins, a complex (NEC: Nuclear Egress Complex) tethered to the INM (Fig. 8). Several transmembrane glycoproteins are encoded by all herpesvirus members and participate in various membrane associated events. We aim at identifying novel factors involved in primary and secondary membrane envelopment using the yeast 2-hybrid system, and validate our findings by various methods, including conventional pull-down experiments, by Lumier, by BRET, and by TAP purification. Functional analysis includes HSV-1 BAC mutagenesis (in collaboration with Prof. Dr. B. Sodeik, MH Hannover and Prof. Dr. U. Koszinowski/Dr. Z. Ruscic, LMU-Genzentrum München), fluorescence and electron microscopy in the course of infection, as well as gene silencing by siRNA.

This project funded by NGFN-Plus (Nationales Genomforschungsnetzwerk) and SFB 576 (Deutsche Forschungsgemeinschaft) focuses on the function of herpesviral and cellular micro RNAs (miRNAs) during the pathogenesis of herpesviruses. MiRNAs are a class of small, single-stranded RNAs involved primarily in the negative regulation of gene expression. MiRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3’ untranslated region (UTR) of specific target mRNAs. Recently it was shown that pathogens like herpesviruses and some other viruses also encode miRNAs (Fig. 9).
We focus on three major questions:
1.)    What are the cellular targets of viral miRNAs?
2.)    What interactions exist between viral proteins and the cellular miRNA   machinery?
3.)    Do the expression patterns of cellular and vial miRNAs change during infection?
For our investigation, we use a plethora of cutting-edge technologies including miRNA Microarray Analysis, stable isotope labeling in cell culture (SILAC), robot-assisted high-throughput yeast-two-hybrid screens, Argonaute-pull-down strategies, High throughput Dual-Luciferase Assays to name but a few examples. We closely collaborate with Sébastien Pfeffer (IBMP Strasbuorg), Päivi Ojala (Biomedicum Helsinki), Gunter Meister (MPI Martinsried), Peter Ghazal (scGTI, Edinburgh) and others.

In this collaborative project with the group of Prof. Juergen Heesemann, Max-von-Pettenkofer Institute (Bacteriology), we perform a genome-wide Y2H Screen to characterise (i) the protein-protein interaction network between Yersinia pYV encoded proteins and host proteins (in collaboration with Dr. Manfred Koegl, DKFZ Heidelberg) and (ii) the protein-protein interaction network of Yersinia pYV encoded proteins (in collaboration with Prof. Peter Uetz & Thorsten Stellberger, Forschungszentrum Karlsruhe) (Fig. 11).
Yersinia enterocolitica is a frequent cause of gastrointestinal disease. It evades the host’s immune response by injecting, via a virulence-plasmid (pYV) encoded Type 3 secretion system, anti host effector proteins (Yops) into the host cell cytoplasm. To generate a protein-protein interaction network, all 74 ORFs of the prototypical virulence plasmid pYVa127/90 plus functional domains of 27 proteins were cloned into yeast-2-hybrid bait and prey vectors (>194 plasmid constructs in total). We screened four human cDNA libraries (bone marrow, testis, lymph nodes, ORF Libraries) for protein-protein interactions with Yersinia pYV proteins using an automatic high-throughput Y2H approach, and also performed Y2H screens to identify new protein-protein interactions between Yersinia pYV proteins. The analysis of interaction partners of individual Yersinia proteins sheds new light on mechanisms of molecular pathogenesis of Yersinia.