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

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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Influence of a Putative Intermolecular Interaction between Core and the Pre-S1 Domain of the Large Envelope Protein on Hepatitis B Virus Secretion

Journal of Virology ◽

10.1128/jvi.76.13.6510-6517.2002 ◽

2002 ◽

Vol 76 (13) ◽

pp. 6510-6517 ◽

Cited By ~ 33

Author(s):

Sophie Le Pogam ◽

Chiaho Shih

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Wild Type ◽

The Core ◽

Virion Release ◽

Naturally Occurring ◽

B Virus ◽

Kinetics Of ◽

Viral Reverse Transcription

ABSTRACT Virion release of hepatitis B virus (HBV) from hepatocytes is a tightly regulated event. It is a dogma that only the mature HBV genome is preferentially allowed to export from the intracellular compartment (J. Summers and W. S. Mason, Cell 29:403-415, 1982). Recently, an “immature secretion” phenotype of a highly frequent naturally occurring HBV variant containing a leucine residue at amino acid 97 of the core protein was identified. Unlike wild-type HBV, this variant secretes almost equal amounts of mature and immature genomes. This phenomenon is not caused by any instability of core particles or by any deficiency in viral reverse transcription (T. T. Yuan, P. C. Tai, and C. Shih, J. Virol. 73:10122-10128, 1999). In this study, our kinetic analysis of virion secretion of the mutant F97L (phenylalanine to leucine) indicates that the secretion of its immature genome does not occur earlier than that of its mature genome. In addition, the secretion kinetics of the mature genomes are comparable between the wild-type HBV and the mutant F97L. Therefore, the immature secretion phenomenon of mutant F97L is not caused by premature secretion or more efficient secretion. Previously, we hypothesized that the immature secretion phenotype is probably caused by the aberrant interaction between its mutant core and wild-type envelope proteins. Here, we further demonstrated that a pre-S1 envelope mutation at position 119, changing an alanine (A) to a phenylalanine (F), can offset the immature secretion phenotype of the mutant I97L (isoleucine to leucine) and successfully restore the wild-type-like selective export of the mature genome of the double mutant pre-S1-A119F/core-I97L.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1010057 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010057

Author(s):

Hui Liu ◽

Junjun Cheng ◽

Usha Viswanathan ◽

Jinhong Chang ◽

Fengmin Lu ◽

...

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Amino Acid Residues ◽

Viral Dna ◽

Viral Dna Replication ◽

Dimer Interface ◽

Rich Domain ◽

B Virus

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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Virion Secretion of Hepatitis B Virus Naturally Occurring Core Antigen Variants

Cells ◽

10.3390/cells10010043 ◽

2020 ◽

Vol 10 (1) ◽

pp. 43

Author(s):

Chiaho Shih ◽

Szu-Yao Wu ◽

Shu-Fan Chou ◽

Ta-Tung Thomas Yuan

Keyword(s):

Amino Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Natural Infection ◽

Antiviral Drug ◽

Core Antigen ◽

Hydrophobic Pocket ◽

Naturally Occurring ◽

B Virus

In natural infection, hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations. The most frequent HBc variant in chronic hepatitis B patients is mutant 97L, changing from an isoleucine or phenylalanine to a leucine (L) at HBc amino acid 97. One dogma in the HBV research field is that wild type HBV secretes predominantly virions containing mature double-stranded DNA genomes. Immature genomes, containing single-stranded RNA or DNA, do not get efficiently secreted until reaching genome maturity. Interestingly, HBc variant 97L does not follow this dogma in virion secretion. Instead, it exhibits an immature secretion phenotype, which preferentially secretes virions containing immature genomes. Other aberrant behaviors in virion secretion were also observed in different naturally occurring HBc variants. A hydrophobic pocket around amino acid 97 was identified by bioinformatics, genetic analysis, and cryo-EM. We postulated that this hydrophobic pocket could mediate the transduction of the genome maturation signal for envelopment from the capsid interior to its surface. Virion morphogenesis must involve interactions between HBc, envelope proteins (HBsAg) and host factors, such as components of ESCRT (endosomal sorting complex required for transport). Immature secretion can be offset by compensatory mutations, occurring at other positions in HBc or HBsAg. Recently, we demonstrated in mice that the persistence of intrahepatic HBV DNA is related to virion secretion regulated by HBV genome maturity. HBV virion secretion could be an antiviral drug target.

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Hepatitis B Virus Capsid Assembly Is Enhanced by Naturally Occurring Mutation F97L

Journal of Virology ◽

10.1128/jvi.78.17.9538-9543.2004 ◽

2004 ◽

Vol 78 (17) ◽

pp. 9538-9543 ◽

Cited By ~ 50

Author(s):

Pablo Ceres ◽

Stephen J. Stray ◽

Adam Zlotnick

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Nucleic Acid Metabolism ◽

Wild Type ◽

Naturally Occurring ◽

Chronic Hepatitis B Virus ◽

B Virus ◽

The Difference

ABSTRACT In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.

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Functional Characterization of Naturally Occurring Variants of Human Hepatitis B Virus Containing the Core Internal Deletion Mutation

Journal of Virology ◽

10.1128/jvi.72.3.2168-2176.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2168-2176 ◽

Cited By ~ 45

Author(s):

Thomas Ta-Tung Yuan ◽

Min-Hui Lin ◽

Sui Min Qiu ◽

Chiaho Shih

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Hbv Infection ◽

Wild Type ◽

Internal Deletion ◽

The Core ◽

Naturally Occurring ◽

Human Hepatitis ◽

B Virus

ABSTRACT Naturally occurring variants of human hepatitis B virus (HBV) containing the core internal deletion (CID) mutation have been found frequently in HBV carriers worldwide. Despite numerous sequence analysis reports of CID variants in patients, in the past decade, CID variants have not been characterized functionally, and thus their biological significance to HBV infection remains unclear. We report here two different CID variants identified from two patients that are replication defective, most likely due to the absence of detectable core protein. In addition, we were unable to detect the presence of the precore protein and e antigen from CID variants. However, the production of polymerase appeared to be normal. The replication defect of the CID variants can be rescued in trans by complementation with wild-type core protein. The rescued CID variant particles, which utilize the wild-type core protein, presumably are enveloped properly since they can be secreted into the medium and band at a position similar to that of mature wild-type Dane particles, as determined by gradient centrifugation analysis. Our results also provide an explanation for the association of CID variants with helper or wild-type HBV in nature. The significance of CID variants in HBV infection and pathogenesis is discussed.

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