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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI034329-05
Application #
2457765
Study Section
Experimental Virology Study Section (EVR)
Project Start
1993-08-01
Project End
1998-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106