INSIGHT INTO POLIOVIRUS GENOME REPLICATION AND ENCAPSIDATION REVEALED FROM MOLECULAR CHARACTERIZATION OF 3B-3C CLEAVAGE SITE MUTANTS

Open Access
Author:
Oh, Hyung Suk
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 22, 2009
Committee Members:
  • Craig Eugene Cameron, Dissertation Advisor
  • Craig Eugene Cameron, Committee Chair
  • Kenneth Charles Keiler, Committee Member
  • B Tracy Nixon, Committee Member
  • Joseph C. Reese, Committee Member
  • Philip C. Bevilacqua, Committee Member
Keywords:
  • cleavage site mutant
  • polyprotein processing
  • replication
  • poliovirus
  • complementation
Abstract:
The picornavirus family consists of nine genera. Many members are important pathogens of human and other animals. Efficient vaccines for many picornaviruses have not been developed. Poliovirus (PV) is one of the extensively studied viruses in this family. Although PV vaccine is available, it is clear that improvements are needed to increase efficiency and safety. In addition, effective antiviral drugs for picornaviruses have not been developed. Therefore, understanding the detailed mechanism of virus multiplication is critical to facilitate rational antiviral drug design and development of more efficient vaccines. PV replication is primed by the 22 amino acid protein, VPg. The PV RNA-dependent-RNA-polymerase, 3Dpol, uridylylates VPg. Recently, the VPg-containing precursor, 3BC, was reported as a more efficient primer in vitro than VPg. However, it has not been determined if 3BC can be used for complete PV RNA synthesis. Poliovirus contains a single positive-strand RNA encoding all structural and non-structural proteins. The entire polyprotein is translated and then processed by virally encoded proteases. This processing is required to produce intermediates and fully-processed viral proteins. Both intermediate and fully-processed proteins are required to facilitate virus reproduction. However, studying functions of individual proteins in the context of the virus life cycle is difficult. Mutagenesis of the cleavage site affects the kinetics of a site’s processing, which results in altered accumulation of specific proteins. Phenotypes induced by cleavage site mutations provide insight into functions of viral proteins. In this thesis, two 3B-3C cleavage site mutants were used to understand functions of proteins in various steps in the PV life cycle. In chapter 3, it is demonstrated that 3BC can be used as a primer for PV RNA synthesis in vivo. This study was performed using the 3B-3C cleavage mutant (Gln-Gly to Gly-Gly: GG) to prevent processing. GG PV replicates in cells. The presence of 3BC-linked full length RNA was confirmed by immunoprecipitation and Northern blotting. This result allowed us to propose a more feasible mechanism for the transition of RNA initiation to elongation during PV replication. The GG mutant is lethal for virus production. Attempting infectious GG PV production gave rise to another mutant virus, EG PV. EG PV is also a 3B-3C cleavage site mutant, but it is viable. In chapter 4, EG PV was characterized. Replication and virus production of EG PV are slower than WT. EG PV alters the kinetics of 3B-3C processing, resulting in a reduced amount of 3CD accumulation. Ectopic expression of 3CD restores the kinetics of replication without improving the kinetics of virus production. Based on this result, a 3CD-trans-complementable step in the PV life cycle is revealed. A virus is a parasite. Viruses have evolved to modify the cellular environment to optimize their reproduction. PV induces vesicles for formation of its replication complexes. Interaction networks between virus and host factors are critical for the vesicle formation. Recently, it was shown that 3A and 3CD can induce relocalization of Arf1 to membranes. In chapter 5, connections between PV replication and cellular modification are revealed. 3CD participation in PV-induced vesicle formation was demonstrated by comparing WT- and EG-PV induced vesicular phenotypes. An Arf1 and 3CD connection to PV-induced vesicle formation was confirmed. Based on these data, a model for 3CD involvement in PV-induced vesicle formation is proposed. Overall, this study provides evidence for a novel function for 3CD in polyprotein processing, RNA replication and encapsidation. Previously proposed but unidentified steps in the PV life cycle are revealed. The relationship between virus and host factors is reinforced. Finally, data presented in this thesis will provide insight into detailed mechanisms of RNA replication and encapsidation in other picornaviruses as well.