Biogenesis of vaccinia: Molecular basis for the hemagglutination-negative phenotype of the IHD-W strain

Abstract

The hemagglutinin negative (HA −) strain IHD-W of vaccinia virus was shown to induce a defective glycopeptide. The serorelatedness between HA and the altered molecule could be demonstrated by use of specific anti-HA antisera. The HA − antigen isolated from immunoprecipitates was analyzed by means of “Western” blots, peptide mapping, using liquid chromatography and antigen competition, in conjunction with radioimmuno assay. Despite the reduced molecular weight (MW) of the polypeptide in the HA − 41K glycoprotein as compared with that of the polypeptide in the normal 85K HA, antigenicity was conserved. Absence of HA activity from the 41K defective molecule was related to the presence of only the N-type glycosidic links between the carbohydrate and asparagine residues in the polypeptide. Following synthesis, the active HA molecule was transfered to the cell surface but the defective glycoprotein accumulated intracellularly, probably in association with the smooth membrane reticulum. This implies that vectorial transfer to the cell surface was defective in the case of the mutant molecule. In vitro translation produced the unglycosylated form of the HA polypeptide, 57K in MW and the HA − polypeptide, 30K in MW. With the latter product there was a 2K difference in MW between the protein synthesized in vitro and the 28K one, formed during in vivo infection in the presence of tunicamycin. This result suggests that reduction in MW may involve cleavage of a peptide, perhaps a “signal” peptide, during formation of the glycoprotein. When considered altogether, these observations indicate that mutation, in the gene encoding the HA resulted in profound changes in molecular structure and cytopathology. The mutation caused changes in both the protein and carbohydrate moieties of the molecule, failure of the nascent glycoproteins to move vectorially to the cell surface or produce hemagglutination and finally changed the cytopathology from that of cell rounding to syncytium formation.