Characterization of poliovirus 2CATPase bound to bilayer nanodiscs and involvement of the poliovirus 3Dpol thumb α-helix in determining poly(A) tail length
Date
2013
Authors
Springer, Courtney Lee, author
Peersen, Olve B., advisor
Ho, P. Shing, committee member
Luger, Karolin, committee member
Kennan, Alan, committee member
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Abstract
Poliovirus (PV) is a small non-enveloped picornavirus with a ≈7.5 kb long single-stranded, positive-sense RNA genome. Upon infection, the RNA is translated to generate a ≈250 kDa polyprotein that is subsequently cleaved into about a dozen fully processed proteins and several functional intermediates. PV replication occurs in large membrane associated complexes involving the "non-structural" P2 and P3 region proteins and two of these proteins, 2CATPase and 3Dpol, are the subjects of this dissertation. Part I of this work is focused on the 2C protein, an AAA+ family ATPase that plays a key role in host cell membrane rearrangements and virion assembly, but the membrane binding characteristics of 2C and its polyprotein precursors have made it difficult to elucidate their exact roles in virus replication. In this work I show that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C and the precursor 2BC and 2BC3AB polyproteins in a membrane bound form. Biochemical analysis shows that the proteins are highly active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Notably, the ATPase activity of the core 2C domain is stimulated ≈60-fold as compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. This data leads to a model wherein the viral replication complex can be assembled with a minimally active form of 2C that then becomes fully activated upon proteolytic cleavage from the adjacent 2B viroporin domain. In Part II of this dissertation, I focus on the role of the viral RNA polymerase, 3Dpol, in maintaining the ≈20-150 nucleotides long 3' poly(A) tail of the viral genome. The length of the tail is important for viral replication and initiation of (-)-strand synthesis, but the means by which the RNA is polyadenylated and how poly(A) tail length is regulated is not well understood. We have identified several mutations in an α-helix of the 3Dpol thumb domain that directly impact poly(A) tail length. Here, I tested the impact of these mutations on reiterative transcription of poly(A), poly(U), and poly(C) templates as well as characterized their effect on 3Dpol initiation, stability, elongation rate, and fidelity. I found that mutations in the thumb have the greatest impact on elongation complex stability and that 3Dpol is able to reiteratively transcribe homopolymeric poly(U) and poly(A), but not poly(C) RNA templates. Interestingly, distinct poly(A) and poly(U) transcripts are generated from 10 nucleotide homopolymers that are 1, 7, or 8 nucleotides longer than the template. Based on these findings, we propose a poly(A) slippage model in which the elongation complex stalls at the end of the homopolymer stretch in the absence of additional nucleotides to promote a single nucleotide slippage. This is followed by a slow structural rearrangement in which 3Dpol slips back to the 3' end of the homopolymer sequence, where it is able to re-transcribe starting from the fifth poly(U) in the template.
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Subject
enzymology
membrane protein
poliovirus
polymerase
virology
ATPase