The Picornaviridae, a family of small positive-strand RNA viruses includes a large number of important human pathogens that cause a wide variety of diseases, including respiratory infections, meningitis, encephalitis, poliomyelitis, myocarditis and hepatitis. Viral RNAs with deletions in the capsid coding region of the viral genome act as replicons and are efficiently translated and replicated in the host cell. Replicons with most of the polyprotein coding sequence deleted can replicate in in vitro reactions containing viral proteins synthesized in trans from a non-replicating helper RNA. In this proposal, molecular genetic techniques are being used to investigate poliovirus RNA replication. A mutagenic analysis of structure-function relationships for viral proteins and RNA sequences, genetic complementation assays in vivo and in vitro, genetic recombination assays and the isolation and characterization of revertant viruses are being used to define the genetic elements that are required for the replication of the viral genome. Complementation assays have been developed that can be used in transfected cells and in RNA replication assays in vitro. Both conditional and nonviable mutations can be characterized in these assays. The recent demonstration that all of the viral proteins required for RNA replication can be synthesized in trans provides a powerful new approach for genetic analysis of RNA replication. The following specific aims are proposed to continue these studies : (1) Further characterize the cis and trans genetic elements in the viral genome using a mutagenic approach and complementation assays in vivo and in vitro, (2) develop RNA recombination assays for use in the in vitro replication assays, (3) identify cis-active sequences in the viral genome that are required for RNA replication, and (4) characterize RNA sequences and structures in the 3'NTR which are required for viral RNA replication, and test the proposed model for generating revertants in the 3'NTR.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Virology Study Section (VR)
Program Officer
Meegan, James M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Florida
Schools of Medicine
United States
Zip Code
Spear, Allyn; Ogram, Sushma A; Morasco, B Joan et al. (2015) Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex. Virology 485:492-501
Ogram, Sushma A; Flanegan, James B (2011) Non-template functions of viral RNA in picornavirus replication. Curr Opin Virol 1:339-46
Spear, Allyn; Sharma, Nidhi; Flanegan, James Bert (2008) Protein-RNA tethering: the role of poly(C) binding protein 2 in poliovirus RNA replication. Virology 374:280-91
Silvestri, Lynn S; Parilla, Jessica M; Morasco, B Joan et al. (2006) Relationship between poliovirus negative-strand RNA synthesis and the length of the 3' poly(A) tail. Virology 345:509-19
Jurgens, Christy K; Barton, David J; Sharma, Nidhi et al. (2006) 2Apro is a multifunctional protein that regulates the stability, translation and replication of poliovirus RNA. Virology 345:346-57
Sharma, Nidhi; O'Donnell, Brian J; Flanegan, James B (2005) 3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation. J Virol 79:3565-77
Jurgens, Christy; Flanegan, James B (2003) Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of p2 proteins. J Virol 77:1075-83
Morasco, B Joan; Sharma, Nidhi; Parilla, Jessica et al. (2003) Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis. J Virol 77:5136-44
Barton, D J; O'Donnell, B J; Flanegan, J B (2001) 5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J 20:1439-48
Barton, D J; Morasco, B J; Flanegan, J B (1999) Translating ribosomes inhibit poliovirus negative-strand RNA synthesis. J Virol 73:10104-12

Showing the most recent 10 out of 14 publications