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Molecular interactions of a plant virus genome with virus-coded and host cell proteins involved in intercellular virus transport: high-resolution imaging of protein-protein and protein-RNA complexes

Project information

Grant agreement ID: INTAS-2001-00045

  • Start date

    1 June 2002

  • End date

    31 May 2004

Funded under:

IC-INTAS

Coordinated by:

Scottish Crop Research Institute

United Kingdom

Objective

Virus-encoded movement proteins (MP) are essential for cell-to-cell translocation of plant viral genomes. It is widely believed that intra- and intercellular trafficking of viral MP and transport RNP non-virion (MP-RNA) or virion (CP-RNA) complexes depend on their interactions with cellular receptors, the MP-binding proteins (MPBPs). A collaboration of SCRI, MSU and HU teams in frame of INTAS project (Reference number: INTAS-96-1683) resulted in identification and isolation of TMV MP binding proteins, which may mediate and regulate TMV MP and/or MP-RNA complexes intra- and intercellular transport. It was found that three tobacco leaf cell wall (CW)-associated proteins (34K, 42K and 50-55K) specifically bind TMV MP. The 34K protein was isolated by MP affinity chromatography (MPAC), microsequenced and identified as pectin methylesterase (PME), a ubiquitous plant enzyme involved in CW growth and regeneration. Presumably PME localized in plant intercellular connections, the plasmodesmata (PD), plays a role of the MP receptor. The full-length gene of PME was cloned and expressed in E. coli and antibodies to PME were obtained. These procedures will be applied to the isolation of other MPBPs from plant cells and their characterization.

Furthermore, because pathogens usually adapt the host cell pathways for their life cycle, MP-MPBP interactions most likely reflect a more general and fundamental biological phenomenon, intercellular communication and trafficking of macromolecules within and between plant cells. Presently, however, very little is known about the cellular MPBPs and their interactions with viral MPs and transport RNP complexes during macromolecular transport.

The proposed interdisciplinary research will start filling this gap by identifying and characterizing the architecture and structure of such complexes by atomic force microscopy (AFM) and Biacore system, and elucidating the changes in the structure of complexes following phosphorylation in relation to their functions.

These include:
Complexes formed by the MPs and host-plant-coded MPBPs.
Purified MPBPs, in particular, the pectin methylesterase (PME);
Complexes formed by MPBPs and "MP-RNA" ribonucleoproteins (RNPs);
Nontranslatable non-virion "MP-RNA" or virion "CP-RNA" RNP-complexes, and the same RNPs converted into a translatable form after phosphorylation of the MP (or CP);
Native and phosphorylated viral genome-linked protein (VPg) exposed on the surface of the potato virus A. A putative complex formed by the virion VPg with cellular proteins.

Comparison of architecture and structure of biomolecular complexes with different types of movement will shed more light on the mechanisms of movement and explain functional differencies between different viral MPs. Structure-function analysis of biomolecular transport complexes will provide both novel information on molecular mechanisms of movement of macromolecules and tools for further studies.

The present project is aimed at joining the efforts of the four teams (SCRI, MSU, HU, CB) specialized in virology, molecular biology and genetic engineering with the abilities of the ATC team highly experienced in AFM, for further investigation of structural and biological properties of macromolecular transport complexes.

Coordinator

Scottish Crop Research Institute

Address

Invergowrie
Dd2 5da Dundee

United Kingdom

Participants (4)

Advanced Technologies Centre

Russia

M.V. Lomonosov Moscow State University

Russia

Russian Academy of Sciences

Russia

University of Helsinki

Finland

Project information

Grant agreement ID: INTAS-2001-00045

  • Start date

    1 June 2002

  • End date

    31 May 2004

Funded under:

IC-INTAS

Coordinated by:

Scottish Crop Research Institute

United Kingdom