Insights into Hepatitis B Virus Replication Gained from Phosphor Imaging of Southern Blots with the Sapphire Imager

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In a recent publication, Ji Xi et al examined the role of the linker region of the Hepatitis B virus (HBV) core protein, a protein essential for viral replication. The C-terminal domain of the core protein contains multiple phosphorylation sites that are dynamically phosphorylated and dephosphorylated by cellular kinases and phosphatases throughout the viral life cycle. The changes in phosphorylation levels alter the protein charge and regulate the interaction of viral genome with the core protein as needed during HBV particle assembly and maturation. The cellular phosphatases involved in this process have not been identified.

The authors identified a consensus binding motif for B56, a regulatory subunit of the cellular phosphatase PP2A, in the linker region of the HBV core protein. To determine whether PP2A is important to dephosphorylation of the core protein, the authors studied the effects of mutations to this consensus sequence and of pharmacologic inhibition of PP2A.

Though no evidence of direct physical interaction between the consensus sequence and PP2A-B56 was found in this publication, mutations to the consensus sequence had dramatic effects on phosphorylation of the core protein and on multiple viral processes including packaging of viral RNA, reverse transcription, secretion of virions, and the amount of covalently closed circular viral DNA (CCC DNA) produced. In addition, pharmacologic inhibition of PP2A affected core protein phosphorylation and CCC DNA levels. The research indicates the linker region plays an important role recruiting PP2A and other factors to modulate phosphorylation of the core protein C-terminal domain and regulate HBV replication.

To study the levels of viral DNA species the researchers conducted Southern blots detected with 32P-labaled probes. Blots were imaged via phosphor imaging with storage phosphor screens being detected and quantified using a Sapphire Biomolecular Imager.

Figure 13 from Xi J, Luckenbaugh L, Hu J (2021). PLoS

Pathog 17(1): e1009230 licensed under CC BY 4.0.

The Sapphire imager provides multiple imaging capabilities in addition to phosphor imaging including white light, multi-channel fluorescence, and chemiluminescence imaging of blots, tissues, microplates, and more. Learn more about the Sapphire imager and how it can support your research here https://azurebiosystems.com/sapphire-biomolecular-imager/.

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