The proposed research will focus on the structure and function of the Large (L) protein of the paramyxovirus family of non-segmented negative-strand RNA viruses. The paramyxoviruses are a diverse group of infectious agents responsible for a variety of medically and economically important diseases of humans and animals. By comparison to the more abundant paramyxovirus proteins, our understanding of the structure and functions of the L protein is incomplete. The 250 kDa L protein is a multifunctional polypeptide which possesses the catalytic sites involved in various steps in viral RNA synthesis. Because L catalytic activities depend on interactions of the L polypeptide with other viral proteins, the first two goals of this research are to identify the domains of L which direct two classes of essential protein-protein interactions. First, a cDNA clone which expresses the paramyxovirus SV5 L polypeptide-will be employed to map the regions of L which direct interactions with the second viral polymerase subunit protein P. Second, cDNA-derived mutant L polypeptides will be assayed for their ability to bind to the genomic nucleocapsid structure and to be incorporated into progeny virions. The mapping of regions of L involved in these two types of protein-protein interactions will be important, because they will provide the first identification of essential structural domains for this extraordinary multifunctional polypeptide. Moreover, these data will set the stage for a rational mutational approach to identifying the catalytic regions of the L protein. As a third goal addressing the functional domains of L, a recently-developed in vivo replication system will be employed to analyze the SV5 genomic sequences which direct L polymerase functions. Specifically, a model cDNA-derived dicistronic viral genome will be used in a mutational analysis of the intercistronic sequences which modulate L protein functions during viral mRNA transcription. The information gained from these experiments on intercistronic sequences will fill a major gap in our understanding of the signals controlling the various L protein activities. Together, these experiments on the L protein are focused on setting the foundation for the future identification of the catalytic domains of this multifunctional polypeptide and of the cis-acting genomic sequences which regulate its enzymatic functions.
