Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

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Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

Journal of Virology ◽

10.1128/jvi.01001-19 ◽

2019 ◽

Vol 93 (22) ◽

Cited By ~ 3

Author(s):

Szu-Yao Wu ◽

Ya-Shu Chang ◽

Tien-Hua Chu ◽

Chiaho Shih

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Mouse Model ◽

Core Protein ◽

Wild Type ◽

Viral Dna ◽

Naturally Occurring ◽

B Virus

ABSTRACT Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild-type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant, I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants, I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: (i) excessive numbers of serum HBV virions containing immature genomes, (ii) significantly reduced numbers of intracellular relaxed-circle and single-stranded DNAs, and (iii) less persistent intrahepatic and secreted HBV DNAs than wild-type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model. IMPORTANCE Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.

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Low-Level Secretion of Human Hepatitis B Virus Virions Caused by Two Independent, Naturally Occurring Mutations (P5T and L60V) in the Capsid Protein

Journal of Virology ◽

10.1128/jvi.74.19.9099-9105.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9099-9105 ◽

Cited By ~ 46

Author(s):

Sophie Le Pogam ◽

Thomas Ta-Tung Yuan ◽

Gautam Kumar Sahu ◽

Soma Chatterjee ◽

Chiaho Shih

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Capsid Protein ◽

Core Protein ◽

Wild Type Virus ◽

Viral Dna ◽

Naturally Occurring ◽

Human Hepatitis ◽

B Virus

ABSTRACT The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the “low-secretion” phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both α-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.

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An adenovirus-delivered peptide aptamer C1-1 targeting the core protein of hepatitis B virus inhibits viral DNA replication and production in vitro and in vivo

Peptides ◽

10.1016/j.peptides.2009.07.006 ◽

2009 ◽

Vol 30 (10) ◽

pp. 1816-1821 ◽

Cited By ~ 11

Author(s):

Wei Zhang ◽

Wei Ke ◽

Si-Si Wu ◽

Lu Gan ◽

Rui Zhou ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Viral Dna ◽

Viral Dna Replication ◽

The Core ◽

B Virus ◽

Peptide Aptamer

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Dominant negative mutants of the duck hepatitis B virus core protein interfere with RNA pregenome packaging and viral DNA synthesis

Hepatology ◽

10.1002/hep.510300139 ◽

1999 ◽

Vol 30 (1) ◽

pp. 308-315 ◽

Cited By ~ 15

Author(s):

Fritz von Weizsäcker ◽

Josef Köck ◽

Stefan Wieland ◽

Wolf-Bernhard Offensperger ◽

Hubert E. Blum

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Core Protein ◽

Dominant Negative ◽

Viral Dna ◽

Duck Hepatitis B Virus ◽

Virus Core ◽

Duck Hepatitis ◽

B Virus

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Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA

Journal of Hepatology ◽

10.1016/j.jhep.2016.09.005 ◽

2017 ◽

Vol 66 (2) ◽

pp. 288-296 ◽

Cited By ~ 33

Author(s):

Kuan-hui Xiang ◽

Eleftherios Michailidis ◽

Hai Ding ◽

Ya-qin Peng ◽

Ming-ze Su ◽

...

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Surface Protein ◽

Amino Acid Substitutions ◽

Viral Dna ◽

Virus Surface ◽

B Virus

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Amino acid residues of core region of hepatitis B virus. Asymptomatic carriers versus patients with liver disease

Journal of Gastroenterology and Hepatology ◽

10.1111/j.1440-1746.1991.tb01481.x ◽

1991 ◽

Vol 6 (3) ◽

pp. 292-296 ◽

Cited By ~ 6

Author(s):

TOSHIKI EHATA ◽

MASAO OMATA ◽

OSAMU YOKOSUKA ◽

KAZUHIKO HOSODA ◽

MASAO OHTO

Keyword(s):

Liver Disease ◽

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Region ◽

Amino Acid Residues ◽

Asymptomatic Carriers ◽

B Virus

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Subtype-Independent Immature Secretion and Subtype-Dependent Replication Deficiency of a Highly Frequent, Naturally Occurring Mutation of Human Hepatitis B Virus Core Antigen

Journal of Virology ◽

10.1128/jvi.73.12.10122-10128.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10122-10128 ◽

Cited By ~ 42

Author(s):

Thomas Ta-Tung Yuan ◽

Pei-Ching Tai ◽

Chiaho Shih

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Hbv Dna ◽

Core Antigen ◽

Wild Type ◽

Virion Assembly ◽

Human Hepatitis ◽

B Virus

ABSTRACT The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype aywmutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtypeadr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, theadr variant I97L replicates as well as its parentaladr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by thetrans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.

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Duck Hepatitis B Virus Nucleocapsids Formed by N-Terminally Extended or C-Terminally Truncated Core Proteins Disintegrate during Viral DNA Maturation

Journal of Virology ◽

10.1128/jvi.72.11.9116-9120.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 9116-9120 ◽

Cited By ~ 27

Author(s):

Josef Köck ◽

Stefan Wieland ◽

Hubert E. Blum ◽

Fritz von Weizsäcker

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Dna Synthesis ◽

Core Protein ◽

Viral Dna ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

Core Proteins ◽

B Virus ◽

Carboxy Terminal

ABSTRACT Hepadnaviruses are DNA viruses that replicate through reverse transcription of an RNA pregenome. Viral DNA synthesis takes place inside viral nucleocapsids, formed by core protein dimers. Previous studies have identified carboxy-terminal truncations of the core protein that affect viral DNA maturation. Here, we describe the effect of small amino-terminal insertions into the duck hepatitis B virus (DHBV) core protein on viral DNA replication. All insertion mutants formed replication-competent nucleocapsids. Elongation of viral DNA, however, appeared to be incomplete. Increasing the number of additional amino acids and introducing negatively charged residues further reduced the observed size of mature viral DNA species. Mutant core proteins did not inhibit the viral polymerase. Instead, viral DNA synthesis destabilized mutant nucleocapsids, rendering mature viral DNA selectively sensitive to nuclease action. Interestingly, the phenotype of two previously described carboxy-terminal DHBV core protein deletion mutants was found to be based on the same mechanism. These data suggest that (i) the amino- as well as the carboxy-terminal portion of the DHBV core protein plays a critical role in nucleocapsid stabilization, and (ii) the hepadnavirus polymerase can perform partial second-strand DNA synthesis in the absence of intact viral nucleocapsids.

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