Group: Viral proteins

Scientific focus:

Main research interests of the group are:
- Structural genomics of human coronaviruses
- Structure and function of coronaviral nonstructural proteins
- Production of new viral protein targets suitable for crystallization and antiviral drug development
Virus targets:

Human coronaviruses (plus-strand RNA viruses):
SARS-CoV, HCoV-229E, HCoV-NL63 and HCoV-HKU-1
Lassa virus (negative-strand RNA virus)
Sapovirus (plus-strand RNA virus)
The genomes of coronaviruses are composed of a single-stranded RNA organized in several open reading frames encoding both structural and nonstructural proteins. The viral RNA is enveloped by the nucleocapsid protein N bound to the viral membrane protein M. The spike protein S is interacting with surface receptors inducing fusion of the virus with the host cell.
Figure 1: Schematic view of coronaviruses
K. V. Holmes and L. Enjuanes (2003), Science 300, 1377-1378
Two polyproteins pp1a and pp1ab are translated from the viral RNA after virus entry. Polyprotein processing by the viral papain-like proteases and the main protease results in 16 nonstructural proteins. Several or all of the nonstructural proteins build the replicase complex mediating polyprotein processing, viral transcription and replication.
Figure 2: Polyprotein processing to mature nonstructural proteins
A. E. Gorbalenya et al. (2004), J. Virol. 78, 7863-7866
Orange arrow heads - cleavage of the polyproteins by papain-like proteases
Blue arrow heads – cleavage of the polyproteins by main protease
The experimental outline in the structural genomics project is a combined strategy of new cloning techniques (recombination cloning), high-throughput screening methods (E-Base HTS electrophoresis) and biophysical procedures (dynamic light scattering, circular dichroism, fluorescence spectroscopy). Intact overproduced viral proteins and protein domains are subjected to X-ray structure determination by Jeroen Mesters´ group immediately after successful crystallization.
Figure 3: Experimental strategy towards crystallization and drug development.
96-sample HTS electrophoresis system (right side) screening E. coli lysates under different induction conditions for overproduction of coronaviral proteins.
Dynamic light scattering (DLS) measures the time dependence of the light scattered from a very small region of solution over a time range from micro- to milliseconds. The fluctuations in the intensities of scattered light are related to the diffusion coefficient of particles by the Stokes-Einstein equation. Data are processed in terms of particle size (hydrodynamic diameter) which are related to the diffusion coefficient. DLS is a valuable method to check the proteins´ aggregation state before crystallization. Detection of aggregates indicate that crystallization will not be successful.
Figure 4: Dynamic (quasi-elastic) light scattering (DLS)
DLS device Spectroscatter 201 (RiNA GmbH Netzwerk RNA Technologies)
Non-structural proteins are prone to polymerization, but disaggregate after adding additives. For instance the human coronaviral Nsp8 proteins display smaller hydrodynamic diameters and monodisperse distributions under reducing and metal-ion chelating conditions favoring successful crystallization
Figure 5: DLS of the nonstructural proteins Nsp8 of SARS coronavirus and human coronavirus HCoV-229E
Fluorophores like 1,8-anilino-naphtalene-sulfonate (1,8-ANS) are used as probes binding non-covalently to hydrophobic pockets in nonstructural proteins. Binding is indicated by a blueshift in the fluorescence spectrum and a strong increase in the quantum yield. Fluorescence comparisons of homologous proteins like the Nsp8s of SARS-CoV and HCoV-229E prove conservation of the 1,8-ANS binding site which might be of functional importance in protein-protein and protein-RNA interactions.
Figure 6: Fluorescence spectroscopy of coronaviral proteins
Red – excitation spectra, blue – emission spectra