Type
Text
Type
Dissertation
Advisor
Konopka, James | Wimmer, Eckard , Carter, Carol | Freimuth, Paul | Grubman, Marvin.
Date
2011-12-01
Keywords
Virology--Molecular biology--Health sciences | 2CATPase, alanine scanning mutagenesis, capsid proteins (VP1 and VP3), encapsidation, poliovirus
Department
Department of Molecular Genetics and Microbiology
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/71434
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Polypeptide 2CATPase is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains, important for uncoating, host cell membrane alterations, RNA replication and encapsidation have previously been identified. In this study, charged to alanine scanning mutagenesis was used to generate conditional-lethal mutations in hitherto uncharacterized domains of the 2CATPase polypeptide, particularly those possibly involved in morphogenesis. Adjacent or clustered charged amino acids (2-4), scattered along the 2CATPase coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, ten lethal, one severely temperature-sensitive, two quasi-infectious, and three wild type-like mutants were identified. Using a Renilla luciferase reporter virus, all lethal and quasi-infectious mutants demonstrated RNA replication defects. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. These mutants have led to the identification of several new sites within the 2CATPase polypeptide that are required for RNA replication. Interestingly, I characterized a quasi-infectious mutant (K6A/K7A) that produced a suppressor mutation (G1R) and a novel 2B^2CATPase cleavage site (Q^R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. Furthermore, analysis of the suppressor mutants of one quasi-infectious mutant and a detailed mutagenic analysis of its flanking Cysteine Rich regions have revealed a new domain near the C-terminus of 2CATPase that is involved in encapsidation possibly achieved through interacting with a spacer between A and B motifs of the NTP-binding domain of 2CATPase. Most importantly, suppressor mutations were identified not only in PV nonstructural protein 2CATPase but also in PV capsid proteins VP3 and VP1 - the first demonstration of genetic suppression of a 2CATPase defect by capsid proteins in the background of PV genome. The data presented here reinforce our previous conclusion that an interaction between 2CATPase and the capsid proteins is required for viral encapsidation. In addition, I performed a yeast two hybrid screening of a HeLa cell cDNA library and identified several cellular factors that may interact with PV non-structural protein 2CATPase. The roles of these candidates in PV replication and/or assembly of PV viral particles, if any, need further investigations. | 199 pages
Recommended Citation
Wang, Chunling, "Functional analysis of poliovirus protein 2CATPase in viral RNA replication and encapsidation using alanine scanning mutagenesis" (2011). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 640.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/640