The elucidation of mechanisms of synthesis, processing and transport of viral components at the molecular level will help us to understand mechanisms that govern biosynthesis of normal cellular components and to understand problems of defective viral assembly implicated in some neurological diseases. To study the biosynthesis of viral macromolecular components, we have prepared a battery of monoclonal antibodies reacting with different sites of polypeptides of two negative stranded RNA viruses (Vesicular stomatitis virus (VSV) and measles virus), polyclonal antibodies made against synthetic peptides corresponding in sequence to portions of the viral polypeptides, and genes coding for some of the viral polypeptides of the Vesicular stomatitis virus cloned into convenient expression vectors. One part of the project concerns the study of the ultimate assembly of viral components at the sites of viral budding. High resolution views are obtained from platinum replicas of the outer and inner side of the plasma membrane of cells infected with VSV. Combining this approach with the immunogold labeling technique for viral proteins, we determine how the nucleocapsid interacts with the viral glycoprotein integrated in the plasma membrane and how the M protein may induce nucleocapsid coiling during viral maturation. Studies with a temperature-sensitive mutant in the M protein of VSV indicate that M protein normal transport to the membrane and normal conformation is necessary for nucleocapsid coiling and viral budding to occur. Another part of the project is to analyze the function of the non-structural protein C of measles virus by microinjection of living cells with specific antibodies into this protein.
