Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain

Abstract

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera.The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/ core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles. Hepatitis B virus (HBV)1 infects more than 300 million people and is a major cause of liver diseases. The HBV belongs to the Hepadnavirus family and is a small (42 nm) enveloped DNA virus, which possesses a 27-nm icosahedral nucleocapsid composed of core protein and a 3.2-kb partially doublestranded, circular genome (1). Although the term "Dane particles" refers to the 42-nm HBV particles (2) and is often used in reference to the complete HBV particles, electron microscopic studies have suggested that the DNA-negative "empty" Dane particles are predominant in sera (3-6). The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail.The HBV genome encodes two core-related open reading frames, precore and core genes (Fig. 1). These are expressed because of two in-frame ATG initiation codons located at the 5Ј end of the genes. The first ATG encodes a 25-kDa protein (p25) containing the 29-amino acid (aa) precore sequence fused to the N terminus of the HBV core antigen (HBcAg). The p25 is directed toward the secretory pathway by a 19-aa signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER), producing a 22-kDa protein. Subsequent proteolytic cleavages within the arginine-rich Cterminal region (34 aa) generate a 17-kDa protein that is secreted as hepatitis B e antigen (HBeAg) (7-10). A heterogeneous population of these precore derivatives has been observed in the sera of patients and is serologically defined as HBeAg (9,11,12). Conversely, the second ATG specifies the 21.5-kDa HBcAg, which assembles into dimers that form the virus capsid (7,9,(13)(14)(15). HBcAg is a 183-residue protein wi...

Correlation between serum hepatitis B virus core‐related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients

“…Therefore, the measurement of intrahepatic HBV DNA and/or HBV cccDNA is important for monitoring the viral status of hepatitis patients [Sung et al, 2005], although these assays involve the physical stress of needle biopsy. Several reports indicate that the level of HBcrAg, which is a complex of HBeAg, HBcAg, and p22cr coding precore/core gene [Kimura et al, 2002[Kimura et al, , 2005, reflects the natural course of viral loads in patients under treatment with nucleos(t)ide analogues, and that the reduction rate of HBcrAg is slower than that of serum J. Med. Virol.…”

“…p22cr is a precore protein from amino acid À28 to at least amino acid 150, containing an uncleaved signal sequence and lacking the C-terminal arginine-rich domain. p22cr is found in empty and HBV DNA negative virus particles; the production of empty particles is not dependent on the formation of HBV DNA [Kimura et al, 2005]. Several reports indicate that the concentration of serum HBcrAg is closely correlated with peripheral HBV DNA in untreated patients [Rokuhara et al, 2003;Tanaka et al, 2006].…”

“…Recently, a new assay was performed for the detection of hepatitis B core-related antigen (HBcrAg) consisting of HBV core antigen (HBcAg), HBeAg, and 22 kDa precore protein (p22cr) coded with precore/core gene [Kimura et al, 2002[Kimura et al, , 2005. p22cr is a precore protein from amino acid À28 to at least amino acid 150, containing an uncleaved signal sequence and lacking the C-terminal arginine-rich domain.…”

Simultaneous quantitation of serum HBV DNA and HBeAg can distinguish between slow and fast viral responses to antiviral therapy in patients with chronic hepatitis B

“…On the other hand, HBcAg, the major protein of the HBV capsid, is translated from HBV pregenomic mRNA 21 . Since HBeAg and HBcAg share a sequence consisting of 149 amino acids, they are collectively called Hepatitis B core-related antigens.…”

“…It would also allow for a comparison of the efficacies of potent antiviral drugs when a large proportion of patients have HBV DNA below the detection threshold 21 . ROKUHARA et al 29 reported that five out of six patients treated with LAM for six to eight months still had detectable levels of core-related antigen.…”