RRhinoviral 2A proteinase:Structure cellular targets effects on host cells

Picornaviral 2A and 3C proteinases are exceptional enzymes which are essential for picornaviral replication. they contain a cysteine at the active site, yet the amino acids surrounding this residue are identical to those normally found in serine proteinases. The 2A proteinase is of special interest. In addition to is role in the processing of the primary viral translation product, it is mainly responsible for the host cell shut-off cleaving directly the initiation factor elF-4G. Consequently cap-dependent initiation of cellular protein synthesis is inhibited and initiation at the picornaviral internal ribosome entry site (IRES) is stimulated. Evidence for roles of 2A in addition to these is becoming available. Plans for future research are as follows: (1) Experiments will be performed to investigate cotranslational cleavage between the P1 and P2 region, the first step in the processing of the viral polyprotein. (2) Comparison of the 2A proteinase cleavage rates of elF-4G in free form and as a complex in elF-4F. This should explain discrepancies between data obtained by the various groups in the past. (3) 2A proteinase of human rhinovirus serotype 2 will be produced in amounts sufficient for X-ray analysis in collaboration with Prof. Michael James in Canada. Small crystals of the wild-type enzyme have been obtained so far. However, the quality of the crystals must be improved. We plan to produce mutant 2A proteinases in which certain cysteine residues are exchanged for Ser or Ala and deletion mutants with N-terminal truncations. (4) To search for 2A proteinase targets other than elF-4G, 2A expression in Saccharomyces cerevisiae will be investigated. Production of the rhinoviral 2A results in a rapid inhibition of cell division, yet the two yeast elF-4G homologues are not cleaved and protein synthesis continues. Identification of cellular targets in yeast will greatly facilitate the search for momologous proteins in higher eukaryotic cells. (5) Human rhinoviruses harbouring genes for temperature-sensitive 2A proteinase will be constructed. Temperature shift experiments will allow the detailed study of the role of the enzyme during viral replication and to define effects on the physiology of the infexted host cell. (6) An inducible system for 2A in mammalian cells will be established to determine directly the effect of 2A proteinase on host cells.

Infections by human rhinoviruses (HRV), also known as "common cold viruses", are among the most frequent viral disease of man. So far about 100 different HRV serotypes have been isolated. Due to the large number of serotypes no vaccination against HRV is possible. Therefore, viral enzymes essential for replication are of interest as potential targets for therapeutics. The first viral enzyme synthesized in the infected cell is the "2A proteinase". Within this project the structure and function of this enzyme has been investigated. About half of the common-colds occuring worldwide are due to infections by HRV. The recurrent infection is due to the large number of different HRV serotypes. So far there is no effective treatment for the disease. Infection by HRV is usually harmless. The classical symptoms are coughing, sneezing, sore throat etc. However, infection by HRV weakens the cellular immune system as shown in collaboration with the group of Prof. Knapp from the Institute of Immunology (University of Vienna). As a consequence, secondary infections by other more dangerous pathogens can occur causing bronchitis, asthma, and pneumonia. HRV are small viruses which contain ribonucleic acid (RNA) as genome. The RNA also serves as mRNA. It is translated into a long polyprotein precursor which is subsequently cleaved into the mature viral proteins by viral enzymes. As the viral capsid does not contain any enzymatic activity, all viral enzymes have to be newly synthesized in the infected cell. The first enzyme to be made is the "2A proteinase". This enzyme is responsible for the first cleavage of the polyprotein precursor as well as for the interference with the metabolism of the host cell. As a consequence, protein synthesis of the host cell is shut-off at an early time of infection and the protein synthesis machinery is switched to the production of viral proteins. In collaboration with the group of Prof. Michael James of the University of Alberta (Edmonton, Canada) the structure of the 2A proteinase of HRV serotype 2 has been determined. As this was the first determination of a 2A proteinase structure, the results were of considerable interest (Petersen et al., 1999, EMBO J. 18, 5463-5475). Based on the structure of the 2A proteinase the molecular mechanism of cleavage of the viral polyprotein precursor as well as its interaction with cellular factors of initiation of protein synthesis can be explained. Subsequent experiments have demonstrated that this interaction results in direct cleavage of the cellular factors by 2A proteinase (Hunt et al., 1999, Virus Res. 62, 119- 128; Liebig et al., 2002, FEBS Lett., in press). At a late time point of the infection cycle the cells are lysed and newly synthesized virus is released. 2A proteinase is also active at this stage. It cleaves a cellular protein which is required for the maintenance of the cellular architecture (Seipelt et al., 2000, J.Biol.Chem. 275, 20084-20089). Whether this cleavage is sufficient for the destruction of the infected cells or whether additional cleavages are required will be the subject of further investigations. The results of this research project have been summarized in a review article which will appear in the fall of 2002 (Kuechler et al., 2002, in Molecular Biology of Picornaviruses, B.L. Semler und E. Wimmer, eds., ASM Press, Washington, DC).