HBx is the smallest gene product
of the Hepatitis B virus (HBV)
and an oncogenic stimulus in chronic infections leading to liver disease.
HBx interacts and interferes with numerous cellular processes, but
its modes of action remain poorly understood. It has been invoked
that HBx employs nucleotide hydrolysis to regulate molecular pathways
or protein–protein interactions. In the present study, we reinvestigate
the (d)NTP hydrolysis of recombinant HBx to explore its potential
as a biochemical probe for antiviral studies. For our investigations,
we employed existing soluble constructs (i.e., GST-HBx, MBP-HBx) and
engineered new fusion proteins (i.e., DsbC-HBx, NusA-HBx), which are
shown to serve as better systems for
in vitro
research.
We performed mutational scanning of the computationally predicted
NTP-binding domain, which includes residues associated with clinical
cases. Steady-state and end-point activity assays, in tandem with
mass-spectrometric analyses, reveal that the observed hydrolysis of
all alleged HBx substrates, ATP, dATP, and GTP, is contingent on the
presence of the GroEL chaperone, which preferentially copurifies as
a contaminant with GST-HBx and MBP-HBx. Collectively, our findings
provide new technical standards for recombinant HBx studies and reveal
that nucleotide hydrolysis is not an operant mechanism by which HBx
contributes to viral HBV carcinogenesis.