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Nano-bio-systèmes

Microscopy of living materials

Responsable:

• Nicolas PAPAGEORGIOU (Délégation CNRS)

 

Objectifs

Imaging of living materials such as cells, viruses, proteins, multi-protein complexes or protein/ADN(ARN) complexes is in plane expansion mostly due to recent technological developments. Les different strategies used for this purpose, are situated in between biology, physics, chemistry, instrumentation, computer science or even mathematics. In the IMN Institute we have undertaken such a interdisciplinary study in collaboration with the Structural Virology team of the “laboratoire d’Architecture et Fonction des Macromolécules Biologiques, AFMB, http://www.afmb.univ-mrs.fr. Techniques 1.Transmition Electronic Microscopy (TEM) combined to negative staining techniques. The TEM available apparatuses are a Hitachi HNAR2000, 300keV as well as a Hitachi HF2000, 200keV. 2.Near Field Microscopy using Atomic Force Microscopy, AFM as well Scanninig Tunneling Microscopy, STM, Veeco Nanotech. Subject Study of the non structural proteins nsp9 and nsp10 of the SARS Cov (Severe Acute Respiratory Syndrome) as well as their complexes with RNA strands. We aim to localize and measure if possible, molecular interactions between proteins and RNA strands.

Fig1: Right: The 3D structure of the nsp9 protein as measured X diffraction (PDB code : 1QZ8). Left : The unit cell of the 3D nsp9 crystal.

The Nsp9 et Nsp10 are implicated in the replication cycle of the viral genome. They present an affinity for RNA strands but their exact function is still unknown. Their 3D structure was already measured by X diffraction techniques. Most precicely we study nsp9 and nsp10 complexes with RNA single strands, synthetic (acid polycytidylic poly C et polyurilidylic poly U) or viral sub-genomic.

Fig2: Left and right : Thick film of nsp9 featuring filaments of nsp9 and aggregates. TEM 300kV, x30000, defocus +800 nm, negative stain 2% Uranyl acetate. Center : Linear juxtaposition of nsp9 unit cells gives rise to the filamentous structures observed.

Corona Viruses (http://www.microbiologybytes.com/virology/Coronaviruses.html )[1], are the largest positive strand RNA (~ 30 000 nucleotides) viruses. Although their very large genome their replication error rate is low. They code for several proteins structural and non structural proteins and their replication mecanisme remains very bad known. The existing data concerning the non structural proteins show a large structural diversity and a unique replication mechanism. The characterization of the various proteins and complexes which participate in the viral replication can permit the development of anti-corona virus drugs as well as the understanding of the replication mecanisme of other emergent or neglected (until now) viruses. [1]Michael M. C. Lai & Kathryn V. Holmes. Coronaviridae: The Viruses and Their Replication. Field's Virology

Fig3: TEM Image of the SARS Corona Virus (micro.msb.le.ac.uk/3035/coronaviruses.html)	Until the emergence of the SARS virus the corona viruses have not been studied extensively due ti their low pathogenesis for humans. However, in the case of the SARS virus the mortality rate can reach 10%. Since the first appearance of SARS, four (4) new corona viruses, pathogenic for humans have been identified. They are responsible for bronchiolitis and the Kawasaki syndrome.
Fig 4: RNA single strands of poly(rC) covered by nsp9, TEM image x30000, defocus +300nm, negative stain Ammonium Molybdate 2%	Figure 5: 3D structure of the nsp10 protein measured by X- diffraction (code PDB: 2G9T)

Figure 6: TEM images of nsp10, x30000, defocus -300 nm, negative stain Ammonium molybdate 2%.