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Genetically altering the thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects on virus replication in cell culture.

ABSTRACT: Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.

SUBMITTER: Tan Z

PROVIDER: S-EPMC3592155 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Genetically altering the thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects on virus replication in cell culture.

Tan Zhenning Z Maguire Megan L ML Loeb Daniel D DD Zlotnick Adam A

Journal of virology 20130102 6

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effe ...[more]

PMID: 23283960

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Project description:The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

Dataset Information

Genetically altering the thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects on virus replication in cell culture.

Publications

Genetically altering the thermodynamics and kinetics of hepatitis B virus capsid assembly has profound effects on virus replication in cell culture.

Similar Datasets

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