Primers used for RT-qPCR.

<div> Hepatitis B virus (HBV) ribonuclease H (RNaseH) inhibitors are a potent class of antivirals that prevent degradation of the viral pregenomic RNA during reverse transcription and block formation of mature HBV DNAs. Development of HBV RNaseH inhibitors is entering advanced preclinical analyses. To ensure the mechanism of action was fully understood, we defined the effects of RNaseH inhibitors on other steps of HBV replication. Some <i>N</i>-hydroxypyridinedione (HPD) HBV RNaseH inhibitors significantly reduced accumulation of capsids in HBV-replicating cells. A representative HPD <b>1466</b>, with a 50% effective concentration against HBV replication of 0.25 µM, decreased capsid and core protein accumulation by 50–90% in HepDES19 and HepG2.2.15 cells. Surprisingly<b>, 1466</b> did not affect pregenomic RNA encapsidation, demonstrating a specific effect on empty capsids. HBV genomic replication was not necessary for <b>1466</b>’s inhibitory effect as it decreased capsid accumulation in cells transfected with replication-deficient mutants blocking pgRNA encapsidation (Δ-bulge), DNA synthesis (YMHA), and RNaseH (D702A) activities. <b>1466</b> also decreased capsid and core protein accumulation in cells transfected with a core protein expression plasmid, indicating that other HBV products are unneeded. <b>1466</b> reduced initial capsid assembly rates in biochemical assembly reactions employing purified core protein (Cp149), demonstrating a specific effect on HBV core protein. We conclude that the bimodal HPD HBV RNaseH inhibitor <b>1466</b> is the prototypic member of a new class of capsid assembly modulators (CAM) that inhibits capsid assembly rather than accelerating it, as all other CAM classes do. We propose that this class be called CAM-I, for <i>CAM-inhibitor</i>. These results lay the foundation for identifying bimodal HBV antivirals targeting the RNaseH and capsid assembly. </div>