Hepatitis B Virus Oncoprotein HBx Is Not an ATPase

[Image: see text] 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.