CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging

Abstract

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.

Protein phosphatase 1 catalyzes HBV core protein dephosphorylation and is co-packaged with viral pregenomic RNA into nucleocapsids

“…Binding of structurally distinct small molecular CpAMs to a hydrophobic “HAP” pocket formed between the inter-dimer interfaces alters the dimer-dimer interaction and results in assembly of morphologically “normal” capsids devoid of pgRNA [ 42 ]. In agreement with the notion that Cp dephosphorylation occurs during pgRNA packaging, we showed that treatment of human or mouse hepatocyte-derived cell lines supporting HBV replication with two different CpAMs, ENAN-34017 or GYH-2-18 ( S9 Fig ) efficiently induced the formation of capsids with slightly faster electrophoretic mobility in native agarose gel and inhibited viral DNA replication ( Fig 7 , particle gel assay), presumably due to inhibition of pgRNA encapsidation [ 35 , 43 ]. As anticipated, CpAM treatment also abolished hypophosphorylated Cp ( Fig 7 , Western blot assay, Input).…”

“…Due to the strong tendency of Cp dimers to self-assemble into empty capsids, pgRNA- and DNA-containing nucelocapsids are usually the minor population of total capsids in HBV replicating cells [ 13 ]. Although the regular agarose gel electrophoresis-based particle gel assay cannot efficiently separate the empty capsids and nucleocapsids, the viral RNA and DNA contents of nucleocapsids can be detected by hybridization with a strand-specific probe [ 25 , 35 ]. As anticipated, expression of Cp alone or Cp with R105A mutant Pol assembled only empty capsids ( Fig 1B ).…”

“…While the specific binding of viral DNA polymerase to the stem-loop (ɛ) structure at the 5’ terminus of pgRNA plays an important role in the selective packaging of pgRNA [ 45 ], how the Pol-pgRNA complex is packaged by Cp dimers to form a nucleocapsid remains to be understood. The observation that alteration of Cp dimer-dimer interaction by CpAM treatment as well as substitutions of single amino acid at Cp dimer-dimer interfaces specifically abrogate the assembly of nucleocapsids, but not empty capsids [ 35 , 43 ], implies that packaging of Pol-pgRNA complexes require unique interactions between Cp dimers. Moreover, our finding that Cp is hyper-phosphorylated in free dimers, but dephosphorylated in pgRNA-containing nucleocapsids, together with the observation that inhibition of PP1 enzymatic activity, or reducing its expression by siRNA significantly inhibited Cp dephosphorylation and pgRNA encapsidation, strongly support the hypothesis that PP1 catalyzed Cp dephosphorylation is required for, and takes place during, nucleocapsid assembly.…”

A New Role for Capsid Assembly Modulators To Target Mature Hepatitis B Virus Capsids and Prevent Virus Infection

“…Electron microscopy was not possible due to low yield and purity of mature capsids (data not shown). In native agarose gel electrophoresis, the mobility of viral capsids is primarily determined by surface charge and mass, and mobility shifts can be considered an indicator of physical or structural changes (21). We found a time-and concentrationdependent mobility shift of mature capsids under HAP_R01 treatment.…”

“…Having isolated mature capsids, we investigated if HAP_R01 would induce structural changes of the capsids that could be detected by a mobility shift in native agarose gel electrophoresis (21). Interestingly, we observed a mobility shift of capsid and HBV-DNA bands already after 1-h HAP_R01 treatment (5,000 nM) ( Fig.…”