The role of the rat cytomegalovirus R78 gene in virus assembly

Abstract

The rat cytomegalovirus (RCMV) R78 gene is a very special gene. This is due to the fact that its sequence is close to that of a G protein-coupled receptor, and still the protein of this gene is not expressed in the membrane of infected cells. Previous research has proven that when this gene is modified, viral replication is less efficient than under normal circumstances, although DNA replication is not altered. The function of the R78 gene still remains unknown. Looking at colocalization of the R78 gene product with other proteins might give new insights on the function of the R78 gene. For colocalization experiments two gene products were chosen, pR55 and pR83. The pR55 protein is a major envelope protein, also called glycoprotein B. This protein is responsible for the entry of the virion into the hostcells. The R83 gene encodes a protein that is part of the tegument proteins that are present between the capsid and the envelope. During assembly of new virions, tegument is placed around the capsid and an envelope is formed. Using antibodies to colour pR78, pR55 and pR83, it is possible to see if pR78 colocalizes with one of these proteins. A rabbit anti-pR78 antibody was already available, but antibodies for the pR55 and pR83 were to be made. Making these antibodies was the main goal of this study. Polyclonal antibody development using the electroporation technique requires correct DNA expression plasmids for the injection of the rats. These were made using standard DNA cloning techniques. These include DNA precipitations, ligations, transformations and transfections. Three different vectors were used in this study for the comparison of efficiency of antibody production. Several expression plasmids are now ready to be used for rat immunisation.