Assessing Hepatitis B Viral Kinetics Using an Inducible Nuclear Pore Blockade

Abstract

Hepatitis B virus (HBV) is the etiological agent of hepatitis B, a global health threat resulting in an estimated 296 million chronic infections worldwide that can lead to liber fibrosis, cirrhosis, and hepatocellular carcinoma. The formation and maintenance of HBV covalently closed circular DNA (cccDNA) is the key mechanism of HBV chronicity. Despite the central role of this process in the viral life-cycle, the kinetics of how quickly cccDNA is produced has yet to be clarified. Here, we use an inducible nuclear pore blockade to first investigate the timing of the import of the viral capsid into the nucleus. Through this, we demonstrate that nuclear import occurs with rapid kinetics, as HBV replication is attenuated by nuclear pore blockade within the first hour of infection. Then, to investigate the maintenance of cccDNA pools, we employ nuclear pore blockade and capsid formation inhibitor GLS4 longitudinally to block the path of intracellular genome recycling. From this, we observe that HBV infection is slightly suppressed over the course of 18 days when the path of capsid recycling is blocked. Additionally, we observe that disrupting capsid formation within 12 hours of infection significantly decreases HBV replication levels, suggesting that early import of newly produced capsids creates a more robust infection. Although recent studies predict stable cccDNA molecules that do not require genome replenishment, our results imply that supplementing cccDNA pools via capsid recycling plays a key role in infection. Together, this work clarifies the staging of capsids early in infection and underscores the role of the capsid recycling pathway in HBV persistence.