Studies on the cell-binding properties of the reovirus [sigma]1 protein

Mammalian reoviruses are ds RNA viruses that are not associated with any specific disease process in man. They can cause disease in mice where the type I (Tl) and type 3 (T3) viruses exhibit markedly different tissue tropism and virulence properties. The different disease patterns of these serotypes are due in large part to the viral cell-attachment protein, σl, specifically targeting the virus to either neuronal cells (T3) or ependymal cells (Tl). The main aim of this project was to locate the cell-binding domain of protein al in order to further characterise the virus-cell attachment process. The Tl and T3 σl proteins were expressed in vitro from cloned DNA copies of the respective SI genes. In vitro translations were carried out in a rabbit reticulocyte lysate system optimised with respect to salt and si RNA concentration to maximise the efficiency of translation. The simRNAs were poorly translated in the lysate (in agreement with in vivo and in vitro studies of other groups). Two different methods were therefore employed to effect an increase in the synthesis of the respective al proteins: (i) Oligonucleotide directed mutagenesis of the cloned Sl-1 gene was utilized to simultaneously improve the weak sequence context of the σl initiation codon and reduce the length of the 5' untranslated (leader) region. The resulting "mutant" transcripts possessed a leader sequence of the same length as those synthesized in vivo, (ii) The inclusion of 2-minopurine in the in vitro translation reactions improved the efficiency with which σI of both serotypes was synthesized. This was due, presumably, to an inhibition of the RNA dependent protein kinase, PI/elF- 2a, which is involved in the control of protein synthesis. A cell-binding assay was developed in which oligomerization of type 3 σI was found to be critical for its ability to bind to monolayers of L-cells. It was found that the formation of oligomeric species required an in vitro translation temperature of 37°C instead of the standard 30°C. Protein al-l was neither able to oligomerize or bind to cells. Specific, saturable binding of σl-3 was demonstrated. These studies also revealed that the Tl and T3 virus probably bind to different receptors on L-cells. Deletion mutants of σl-3 were created by oligonucleotide directed mutagenesis as a means of locating the cell-binding domain of this protein. None of the mutant proteins were found to be capable of binding to L-cells. However, a conformational effect could not be discounted as the primary reason for their inability to bind, as opposed to the removal of a critical binding domain