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Research Group Prof. Dr. rer. nat. Karl-Klaus Conzelmann

MOLECULAR BIOLOGY OF NEGATIVE STRAND RNA VIRUSES

 

Areas of investigation
Viruses are the experts in cell biology. Relying on a living cell for their own propagation and evolution, they have found ways to sneak into cells, switch off alarm systems, re-program cell gene expression and protein functions to make cells virus factories. We are studying negative strand RNA viruses (Mononegavirales) including the neurotropic rabies virus (Rhabdoviridae) and the haematotropic measles virus (Paramyxoviridae) to learn how they exploit the cellular machineries and how they trick the host immune system.
A key technology in the laboratory is the genetic engineering of these RNA viruses on cDNA level (reverse genetics). Recombinant rhabdo- and paramyxoviruses with defined gene defects or mutations are being used to reveal the contribution of individual virus proteins to virus replication and to virus-host cell interplay. We are using a broad spectrum of up-date cell biological and biochemical methods, including NGS, mass spectrometry, cell and virus imaging, genome editing, and high content screening. Knowledge of the mechanisms involved not only tells us how cellular systems function and can be therapeutically manipulated but also provides means to re-program the viruses in order to exploit them as biomedical tools or vaccines, and to reveal targets for antiviral therapies.

Introduction

What is rabies virus?

Rabies (Tollwut- rage - rabbia - lyssa -  狂犬病) is a zoonosis, and among the longest known and feared infectious diseases for humans and animals. Though highly effective vaccines and post-exposure treatment are available, rabies still causes more than 55,000 human deaths per year, mostly children in countries with poor infrastructure. In contrast, an effective rabies therapy is not available, and once symptoms appear, patients will die.

Rabies virus pathogenesis

Although progress has been made in the past decades in the fields of molecular biology and epidemiology of RABV, rabies disease and pathogenesis is still enigmatic. RABV has developed a variety of traits to reach and replicate in the CNS. These include highly effective axonal transport mechanisms, exclusive transsynaptic spread, tools to dampen innate immune responses and means to prevent premature neuronal damage. Death by rabies seems not to be due to extensive virus- or inflammation-mediated destruction of neurons as typically observed in viral encephalitis, rather than to neuronal dysfunction, modulation of neuronal activity, and disturbance of essential neuronal circuits. The identification of target molecules for rational therapy of this outrageously neglected disease of poverty is of utmost priority.

Cell biology and biochemistry of rabies virus.

RABV has entirely adapted to growth in the nervous system and in polarized neurons, however, wildtype virus isolates (so called street viruses) can be readily adapted to conventional cell culture including popular cell lines like mouse N2A, hamster BSR, or human HEK293 cells, without losing their specific neurotropism.  The broad permissivity of cell lines for RABV has in the past greatly facilitated studies on RABV molecular biology and biochemistry, including the development of a reverse genetics system, which we have pioneered. By engineering and analyzing recombinant RABV our lab has in the past contributed seminal work to the fields of virus replication, gene expression, assembly, and virus/host cell interplay.

Research Topics

1. Rabies virus neuron-biology

Left: In vitro differentiated GABA-ergic neurons stained with antibodies recognizing the neuronal marker beta-III tubulin. Right: Co-staining of axonal marker Tau (red) and RABV protein (green). Nuclei are visualized with To-Pro3 (blue). ©Max Eizinger

While conventional cell culture is greatly facilitating studies on basic aspects of the virus’ life cycle, including transcription, replication, virus assembly and budding, studies in polarized neurons are required to reveal the typical and specific traits of RABV. Using conventional cell culture, the RABV matrix protein M was identified in our lab as responsible for shaping and budding of virus particles and as a regulator of viral transcription and replication. The RABV glycoprotein G was identified as dispensable for virus assembly and budding, but essential for infectivity, axonal transport, and transsynaptic transmission.
To extend our studies to neurons, we have recently established protocols for feeder-free culture of mouse embryonic stem cells (mESC) and in vitro differentiation to neurons. Using labeled virions or fluorescent protein-expressing viruses we are studying the mechanisms behind the exclusive entry of RABV at axon terminals, retrograde transport in axons, and the exclusive transsynaptic transmission of RABV. Candidate host genes involved are edited by the CRISPR/Cas9 system either in neuronal precursor cells or differentiated neurons.
In addition, we aim at revealing the “neuroprotective” mechanisms of RABV infection as well as the interference with cellular signaling, leading to neuropathogenesis. Unlike most viruses, infection of neurons with RABV does not cause rapid and overt cell damage, such that neurons remain viable and functional for weeks. Individual street and vaccine viruses display a great range in their ability to protect from neuronal damage. We are using clones derived from vaccine and street viruses, chimeric recombinant viruses, and isogenic viruses carrying single and double mutations to address the contribution of individual virus proteins to neuronal signaling, dysfunction, or death. These studies involve a broad range of up-to-date high-content technologies, bioinformatics, and genome editing, and are relevant with respect to the understanding of neurodegenerative diseases in general.
People involved: Dr. Chloé Scordel, M.Sc. Max Eizinger

2. Rabies trans-synaptic tracing and optogenetics

RABVΔG-eGFP mono-transsynaptic tracing of neurons connecting directly to a single starter cell (yellow) in the cortex (from Wertz et al., Science 2015).

Our initial observation that the RABV G protein is dispensable for virus budding and can be replaced by other viral glycoproteins (by pseudotyping or exchange of genes), but is essential for transsynaptic spread, provided the basis for the establishment of the first viral mono-transsynaptic tracing system (Wickersham et al., Neuron 2007). Pseudotyped delta G rabies viruses (ΔG RABV) have emerged as gold standard for mapping of direct synaptic connections and analysis of neuronal circuits in the central and peripheral nervous system, which is a fundamental pillar of modern neuroscience. In combination with optogenetics and modern in vivo imaging methods such tracers are opening entirely new avenues of investigation in neuroscience and help in answering major outstanding questions of connectivity and function of the nervous system (Ghanem and Conzelmann, 2016). We are constantly developing and producing novel tracers for collaborating neurobiologists, particularly in the frame of the SFB 870 (Neuronal circuits).
People involved: Dr. Alexander Ghanem, B. Sc. Verena Pfaffinger, BTA Melanie Nurtsch

3. Innate immune response to RNA viruses.

Co-evolution of viruses with their hosts has led to a highly complex and powerful immune system for discrimination of self- and non-self, or harmless and dangerous on the one hand, and elaborate mechanisms regulating recognition and immune responses, on the other. RNA viruses are sensed predominantly by RNA pattern recognition receptors like RIG-I and TLRs. We are studying the exact nature of the RNA PAMPs of rhabdo- and paramyxoviruses (Runge et al., 2016), how their recognition is prevented, e.g. by shielding the viral RNA in an RNP complex, and how the immune response is downregulated by viral proteins. Particularly early cytokines such as type I and III interferons (IFNs), which integrate innate and adaptive immune responses, are essential targets for viruses.
Viruses are facing pretty different challenges, depending on their tropism. Two models are currently under investigation, namely the neurotropic rabies virus, and measles virus, which replicates in blood cells, which are sentinels for PAMPs and have specialized pathways to mount a vigorous immune response. Accordingly, measles virus is armed with several “immune escape” proteins (P, V, C) able to interfere with multiple pathways leading to interferon and inflammatory cytokine expression. This includes for example the pDC specific TLR9/MyD88- pathway, and NF-kB signaling, which are targeted by the V protein, as we could show recently (Sparrer et al., 2012). RNAseq and bioinformatics is being used to decipher the multiple effects of measles virus infection and single protein expression on the host transcriptome. Rabies virus in contrast relies on a single P protein specialized in preventing interferon induction by IRF3 and JAK/STAT-mediated ISG expression, but not NF-kB induction (reviewed by Rieder and Conzelmann, 2011). Current studies are deciphering the molecular mechanisms behind this very potent immune suppression. Studying the molecular mechanisms behind, we can learn from viruses how the immune system can be activated (by virus-like RNA PAMPs) and how it can be suppressed (by viral antagonist-like factors).
People involved: M.Sc. Marco Wachowius, M.Sc. Alexandru Hennrich

Interference with interferon induction by RABV (left) and measles virus proteins (right)

Publications

To view more publications of the group please follow this link to PubMed.

Current Group Members

Prof. Dr. Karl-Klaus Conzelmann, PI
Email: conzelmann(at)genzentrum.lmu.de
Phone: +49 89-2180-76851

Maximilian Eizinger, PhD
Email: eizinger(at)genzentrum.lmu.de
Phone: +49 89-2180-76850

Alexandru Hennrich, M.Sc., PhD student
Email: hennrich(at)genzentrum.lmu.de
Phone: +49 89-2180-76859

Martina Oberhuber, PhD
Email: oberhuber(at)mvp.uni-muenchen.de

Verena Pfaffinger, M.Sc.
Email: pfaffinger(at)genzentrum.lmu.de
Phone: +49 89-2180-76850

Chloé Scordel, PhD
EMail: scordel(at)genzentrum.lmu.de
Phone: +49 89-2180-76854

Recent Members

Marco Wachowius (-2017)
Dr. Daniel Aberle (-2015)
Dr. Konstantin Sparrer (-2014)
Dr. Kerstin Schuhmann (-2013)
Dr. Martina Rieder  (-2012)
Dr. Christian Pfaller (-2010)

Awards, honours, professional activities

  • Loeffler-Frosch-Prize 1995, GfV, Society for Virology
  • Editorial Board of J.Virology
  • Editorial Board of Virus Res.

Contact

Prof. Dr. Karl-Klaus Conzelmann
Max von Pettenkofer-Institute & Gene Center
Feodor-Lynen-Str. 25
81377 München, Germany
Tel. +49 89 2180-76851
Email: conzelmann(at)genzentrum.lmu.de

Our lab is located in the Gene Center, Campus Großhadern

For travel instructions please see our homepage at the Gene Center