Human Norovirus is a serious health problem that is understudied due to a lack of tractable rescue systems for understanding virus biology. Since its discovery nearly half a century ago, Norovirus has proven a difficult virus to study because pure virus alone does not replicate in any known cell culture system, there is no physiological small animal model, and there exists only inefficient rescue systems for the virus. The lack of a model system in which to study the virus has lead to a paucity of understanding of even basic features of the virus life cycle, general biology, and pathogenesis in humans. The goal of this research endeavor was to adapt the published rescue systems for Norovirus to the clinically relevant GII.4 New Orleans genotype, to increase precision of viral transcripts through the use of ribozymes, and to increase protein expression through human codon optimization, all with the aim of increasing the likelihood of successful recovery of infectious human Norovirus.
A cDNA clone encoding a full-length genome for GII.4 human Norovirus was cloned under control of a T7 promoter sequence. This clone featured a number of modifications upon the published rescue systems for GII.3 Norovirus, including a 5' ribozyme sequence between the T7 promoter and the 5' UTR of the Norovirus genome, as well as a longer poly-A tail corresponding to the natural poly-A tail of human Norovirus (HuNoV). In addition to this base genome, another construct was assembled featuring GII.4 HuNoV cDNA optimized for protein expression in human cells. This optimization relies on a concept known as codon bias, or the genomic frequency of certain codons for a given amino acid. All reading frames of the HuNoV genome in this construct were modified to match human codon bias to increase viral protein expression in an attempt to improve viral rescue efficiency in mammalian cells.
These modifications did not result in measurable protein expression, and neither RNA replication nor viral protein expression was detected in cells transfected with either construct. This thesis demonstrates that GII.4 Norovirus rescue is not dependent on codon optimization, 5' ribozyme sequence, or poly-A tail length.
Table of Contents
Table of Contents
Chapter 1: Introduction - An Overview of Human Norovirus
Host Immune Response
Surrogate Virus Models
Chapter 2: Adapting Published Rescue Systems to GII.4 New Orleans Human Norovirus Using 5' Ribozyme Sequence, Increased Poly-A Tail Length, and Human Codon Optimization
Chapter 3: Summary and Future Directions
Figures and Tables
Figure 1: Design for construct to rescue GII.4 New Orleans HuNoV
Figure 2: Alternative construct for probing the effect of virus codon optimization on translation efficacy and virus life cycle
Figure 3. RFLP analysis of pKBS6-HuNoV and pKBS6-hOpt-HuNoV
Figure 4: In vitro transcription of pKBS6-HuNoV yields RNA of correct length
Figure 5: Viral RNA is detectable in transfected cells
Figure 6: Norovirus VP1 protein is not expressed in transfected cells
Figure 7. Reporter construct design to probe ORF1 translation
Figure 8: Fluorescence is not detected in BSR.T7/5 cells transfected with pKBS6-K-HuNoV.
About this Master's Thesis
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|A Rescue System for GII.4 New Orleans Human Norovirus is Not Dependent on 5' Ribozyme Sequence, Poly-A Tail Length, or Human Codon Optimization ()||2018-08-28||