The elucidation of mechanisms of synthesis, processing and transport of viral components at the molecular level will help us to understand problems of defective viral assembly implicated in some neurological diseases. To study the biosynthesis of viral macromolecular components, we prepared 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 either cloned into convenient expression vectors or labeled to detect viral mRNAs in cells by in situ hybridization. For studies of viral assembly, high resolution views were obtained from platinum replicas of the outer and inner side of the plasma membrane of cells infected with measles and VSV. Combining this approach with EM immunolabeling techniques for viral proteins, we determined 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 are necessary for nucleocapsid coiling and viral budding to occur. Cellular fractionation studies and gel analysis show that, at nonpermissive temperatures, abnormal M proteins accumulate around the perinuclear membranes. The function of the nonstructural protein C of measles virus was analyzed in infected cells microinjected with specific antibodies to this protein. C protein which is associated with viral nucleocapsids inside the cell may be regulating the level of transcription of the other viral proteins.
