Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment.

Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy.

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix.

Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA.

Characterization of Intracellular Precore-Derived Proteins and Their Functions in Hepatitis B Virus-Infected Human Hepatocytes.

Hepatitis B virus (HBV) precore protein is not essential for viral replication but is thought to facilitate chronic infection. In addition to the secreted precore products, including the hepatitis B e antigen (HBeAg) and PreC protein, intracellular precore-derived proteins in HBV-infected human hepatocytes remain poorly characterized, and their roles, if any, remain largely unknown. Here, we detected multiple precore derivatives, including the nonprocessed precursor p25 and the processing intermediate p22, in HBV-infected human hepatocytes as well as human hepatoma cells overexpressing the HBV precore protein.

Cryo-EM structures of human hepatitis B and woodchuck hepatitis virus small spherical subviral particles.

The loss of detectable hepatitis B surface antigen (HBsAg) is considered a functional cure in chronic hepatitis B. Naturally, HBsAg can be incorporated into the virion envelope or assembled into subviral particles (SVPs) with lipid from host cells. Until now, there has been no detailed structure of HBsAg, and the published SVP structures are controversial.

Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons.

Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection.

Host cell-dependent late entry step as determinant of hepatitis B virus infection.

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV.

Toll-Like Receptor 7 Agonist RG7854 Mediates Therapeutic Efficacy and Seroconversion in Woodchucks With Chronic Hepatitis B.

Conventional treatment of chronic hepatitis B (CHB) is rarely curative due to the immunotolerant status of patients. RG7854 is an oral double prodrug of a toll-like receptor 7 (TLR7) agonist that is developed for the treatment of CHB. The therapeutic efficacy, host immune response, and safety of RG7854 were evaluated in the woodchuck model of CHB.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

Background And Aims: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints.

Approach And Results: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e.

Characterization of Hepatitis B Precore/Core-Related Antigens.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide.

Efficacy of an Inhibitor of Hepatitis B Virus Expression in Combination With Entecavir and Interferon-α in Woodchucks Chronically Infected With Woodchuck Hepatitis Virus.

RG7834 is a small-molecule inhibitor of hepatitis B virus (HBV) gene expression that significantly reduces the levels of hepatitis B surface antigen (HBsAg) and HBV DNA in a humanized liver HBV mouse model. In the current study, we evaluated the potency of RG7834 in the woodchuck model of chronic HBV infection, alone and in combination with entecavir (ETV) and/or woodchuck interferon-α (wIFN-α). RG7834 reduced woodchuck hepatitis virus (WHV) surface antigen (WHsAg) by a mean of 2.

Serum HBV RNA composition dynamics as a marker for intrahepatic HBV cccDNA turnover.

Current therapies can suppress the replication of hepatitis B virus (HBV) but cannot clear chronic HBV infection, which afflicts hundreds of millions worldwide. HBV persistence is sustained by the viral covalently closed circular DNA (cccDNA), an episome in the nucleus of infected hepatocytes. cccDNA is refractory to current therapies and its clearance is the holy grail for HBV cure.

Induction of pro-viral grass carp Ctenopharyngodon idella Hsp70 instead of Hsc70 during infection of grass carp reovirus.

Grass carp Ctenopharyngodon idella Hsp70 has been identified to play a functional role in viral attachment of type III grass carp reovirus, GCRV-104. However, it remains to be clarified whether Hsc70, sharing 86% identity with Hsp70, plays a similar role during viral infection. In this study, grass carp Hsp70 was shown to be induced by GCRV-104 in different grass carp cell lines, whereas Hsc70 was expressed in a relatively constant level during the infection.

Identification and characterization of a type I interferon induced by cyprinid herpesvirus 2 infection in crucian carp Carassius auratus gibelio.

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc).

Epigenetic regulation of hepatitis B virus covalently closed circular DNA: Implications for epigenetic therapy against chronic hepatitis B.

Hepatitis B virus (HBV) infection represents a significant public health burden worldwide. Although current therapeutics manage to control the disease progression, lifelong treatment and surveillance are required because drug resistance develops during treatment and reactivations frequently occur following medication cessation. Thus, the occurrence of hepatocellular carcinoma is decreased, but not eliminated.