Charakterisierung des IBV-Spike-Proteins

Infectious bronchitis virus (IBV) is the prototype Coronavirus. Infection occurs mainly in chickens of all ages. Infected chickens show respiratory symptoms and damage of the reproductive tract. Some strains have a predilection for the kidney resulting in nephritis and nephrosis. Although vaccination strategies are applied worldwide, IBV infection still causes problems because of the appearence of new strains and variants. Infectious bronchitis is economically one of the most important poultry diseases. While for several other coronaviruses cellular receptors are already known, no surface molecule has been identified yet, that enables binding of IBV particles. The binding of coronaviruses to the host cell is mediated by their spike proteins. The IBV spike protein shows sialic acid binding activity; it induces haemagglutination by binding to sialic acids on the surface of erythrocytes. Desialylation experiments showed, that sialic acids play an important role during infection of cultered cells with IBV. After removal of sialic acids from the cell surface by neuraminidase treatment, the cells become resistant to an IBV infection. This could be demonstrated with two IBV strains and three different cell llines. Removal of only the a2,3 linked sialic acids turned out to be sufficient, to abolish an IBV Infection. This finding allows the conclusion that a2,3 linked sialic acids serve as receptor determinant for IBV on cultured cells. However, the affinity of IBV to sialoglycoconjugates is significantly weaker than those of influenza A virus or Sendai virus. To characterise the binding site on the spike protein with virial pseudotypes in future experiments , it is important that the S protein is expressed on the cell surface of transfected cells. the intracellular localisation of the S protein was analysed. Expression of the S protein in two different cell lines with the plasmids pTM1 or pCG1 demonstrated, that the IBV spike protein is retained at an intracellular compartment. Analysis of the sequence of the cytoplasmic tail by S protein mutants, showed that the second tyrosine within the sequence YYTTF is essential for the intracellular retention of the spike protein. If this tyrosine is replaced by an alanine, the S protein is transported to the cell surface. A function of this motif as a endocytosis signal could be excluded. Colocalisation studies of the S protein with marker proteins of endoplasmatic reticulum, ER-Golgi-intermediate compartement (ERGIC) and Golgi apparatus showed some colocalisation with all three marker proteins. The clearest colocalisation was seen with the marker protein of the Golgi apparatus. The results of expression experiments show that the cellular retention maschinery  retains the S protein in the Golgi apparatus.