Recombinant expression and bioinformatic analysis of the Hepatitis B virus X protein
Date
2012-09-18
Authors
Thompson, Liam Jed
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Abstract
There are an estimated 350 million people chronically infected with Hepatitis B Virus (HBV), of which
approximately 600 000 die each year from HBV complications including cirrhosis and liver cancer.
The X protein from HBV (HBx) has been implicated in the progression of chronic HBV to liver cancer
and has been reported to manipulate several critical cellular pathways. These include the cell cycle,
the tumour suppressor protein p53, protein degradation and signal transduction pathways. The role
of these interactions in HBV replication and the viral lifecycle is currently unknown. The lack of
animal models and infectable cell lines together with solubility and stability issues related to the
HBx protein have made progress difficult. The reliance on approximate cellular and animal models
has yielded many discordant studies that have confounded our interpretations of the role of HBx.
There have been no novel approaches attempting to express HBx at a quantity and quality sufficient
for high resolution X-ray and nuclear magnetic resonance structural determination. Additionally no
bioinformatic analyses have been applied to HBx, and thus distinctive features of HBx that may be
responsible for these challenges have not been reported.
This thesis describes the detailed experimentation to express and purify HBx in a functional, soluble
and stable form. The study focussed on Saccharomyces cerevisiae and Semliki Forest Virus
(SFV) expression systems, together with the use of a solubility-enhancing Maltose Binding Protein
protein tag (MBP). The S. cerevisiae-based pYES2 and YEp and mammalian expression vectors
showed production of HBx protein. However HBx that had been expressed using S. cerevisiae and
human cells could not be reliably detected in Western blots using antibodies raised against E. coliexpressed
HBx. This result was despite the positive visualisation of HBx using the same antibodies
and immunofluorescence microscopy. This validated previous reports describing the variable antigenicity
of HBx. Furthermore these findings supported the decision to develop eukaryotic-based
HBx expression vectors as results suggested structural differences between eukaryote and prokaryote
expressed protein. HBx was subsequently detected and purified in a soluble and active form
using an MBP tag as well as a SFV expression vector. All of these options provide an excellent point
from which further work at optimising HBx expression and structural elucidation can occur.
Bioinformatic analysis of HBx suggested the presence of protein disorder and protease sensitive
sites within the negative regulatory domain of HBx. Literature descriptions of the molecular promiscuity that protein disorder allows, offers an explanation for the presence of the discordant findings on
HBx interactions and functions. It is generally accepted that proteins containing disorder are tightly
regulated and thus experimental systems employing overexpression methodologies may encourage
cellular toxicity and non-specific interactions through the use of short linear motifs. Evolutionary
analysis of HBx sequences revealed that the eight HBV genotypes (A-H) showed concordance regarding
synonymous and non-synonymous substitutions at the overlapping and non-overlapping
domains of hbx. Substitutions in hbx were most common at positions where a synonymous substitution
occurred in the overlapping partner gene. The presence of sites under positive, neutral and
negative selection were identified across the length of HBx. The different genotypes showed positive
selection indicating selective pressures unique to each, thus offering a contributing explanation for
the variable disease severity observed between the subtypes.
Overall, this thesis has provided novel methods to express and purify HBx in S. cerevisiae and
mammalian cells. These methods, together with an increased understanding of the nature of HBx
sequences through bioinformatic analysis, pave the way to conduct both structural studies and biological
assays to elucidate the genuine roles of HBx in the HBV lifecycle and its contribution to the
progression to liver cancer.