The Hepatitis B Virus Envelope Proteins: Molecular Gymnastics Throughout the Viral Life Cycle

New hepatitis B virions released from infected hepatocytes are the result of an intricate maturation process that starts with the formation of the nucleocapsid providing a confined space where the viral DNA genome is synthesized via reverse transcription. Virion assembly is finalized by the enclosure of the icosahedral nucleocapsid within a heterogeneous envelope. The latter contains integral membrane proteins of three sizes, collectively known as hepatitis B surface antigen, and adopts multiple conformations in the course of the viral life cycle. The nucleocapsid conformation depends on the reverse transcription status of the genome, which in turn controls nucleocapsid interaction with the envelope proteins for virus exit. In addition, after secretion the virions undergo a distinct maturation step during which a topological switch of the large envelope protein confers infectivity. Here we review molecular determinants for envelopment and models that postulate molecular signals encoded in the capsid scaffold conducive or adverse to the recruitment of envelope proteins.

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Assembly of Hepatitis B Virus Envelope Proteins onto a Lentivirus Pseudotype That Infects Primary Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.00959-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10897-10904 ◽

Cited By ~ 15

Author(s):

Ning Chai ◽

Ho Eun Chang ◽

Emmanuelle Nicolas ◽

Severin Gudima ◽

Jinhong Chang ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Lipid Membrane ◽

Hepatitis B Surface Antigen ◽

Envelope Proteins ◽

Human Hepatocytes ◽

Specific Gene ◽

Virus Envelope ◽

Primary Human Hepatocytes ◽

B Virus

ABSTRACT This study demonstrates that the envelope proteins of hepatitis B virus (HBV) could be incorporated into the lipid membrane of lentivirus pseudotype particles. The assembly procedure was initiated by the transfection of 293T cells with three plasmids: (i) a human immunodeficiency virus (HIV) packaging construct, (ii) a transfer plasmid expressing a reporter gene, and (iii) a plasmid expressing large (L), middle (M), and small (S) HBV envelope proteins. After 2 days, hepatitis B surface antigen and the antigenic forms of L, M, and S were detected at the cell surface by flow cytometry. Also, virus particles that were able to infect cultured primary human hepatocytes (PHH) were released. Under optimal conditions, 50% of PHH could be infected. In addition, the susceptibility of PHH and the resistance of other cell types to the pseudotype particles were similar to those observed for HBV and hepatitis delta virus (HDV), which shares the same L, M, and S. Furthermore, the infection of PHH by the pseudotype was sensitive to known inhibitors of HBV and HDV entry. These findings of specific and efficient infection of hepatocytes could be applicable to liver-specific gene therapy and may help clarify the attachment and entry mechanism used by HBV and HDV.

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The RNA binding proteins YTHDC1 and FMRP regulate the nuclear export of N6-methyladenosine modified Hepatitis B Virus transcripts and affect the viral life cycle

Journal of Virology ◽

10.1128/jvi.00097-21 ◽

2021 ◽

Author(s):

Geon-Woo Kim ◽

Hasan Imam ◽

Aleem Siddiqui

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Viral Life Cycle ◽

Circular Dna ◽

B Virus

YTHDC1 and fragile X mental retardation protein (FMRP) bind N6-methyladenosine (m6A) modified RNAs and facilitate their transport to the cytoplasm. Here, we investigated the role of these proteins in Hepatitis B virus (HBV) gene expression and life cycle. We have previously reported that HBV transcripts are m6A-methylated and this modification regulates the viral life cycle. HBV is particularly interesting as its DNA genome upon transcription gives rise to a pregenomic RNA (pgRNA), which serves as a template for reverse transcription to produce the relaxed circular DNA that transforms into a covalently closed circular DNA (cccDNA). While m6A modification negatively affects RNA stability and translation of viral transcripts, our current results revealed the possibility that it may positively affect pgRNA encapsidation in the cytoplasm. Thus, it plays a differential dual role in the viral life cycle. YTHDC1 as well as FMRP recognize m6A-methylated HBV transcripts and facilitate their transport to the cytoplasm. In cells depleted with YTHDC1 or FMRP, viral transcripts accumulate in the nucleus to affect the viral life cycle. Most importantly, the core-associated DNA and subsequent cccDNA syntheses are dramatically affected in FMRP or YTHDC1-silenced cells. This study highlights the functional relevance of YTHDC1 and FMRP in the HBV life cycle with the potential to arrest liver disease pathogenesis. IMPORTANCE YTHDC1 and FMRP have been recently implicated in the nuclear export of m6A modified mRNAs. Here, we show that FMRP and YTHDC1 proteins bind with m6A-modified HBV transcripts and facilitate their nuclear export. In the absence of FMRP and YTHDC1, HBV transcripts accumulate inside the nucleus to reduce reverse transcription inside HBV core particles and subsequently the cccDNA synthesis. Our study shows how m6A binding proteins can regulate the HBV life cycle by facilitating the nuclear export of m6A-modified HBV RNA.

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Retroviral Restriction Factors Fv1 and TRIM5α Act Independently and Can Compete for Incoming Virus before Reverse Transcription

Journal of Virology ◽

10.1128/jvi.80.5.2100-2105.2006 ◽

2006 ◽

Vol 80 (5) ◽

pp. 2100-2105 ◽

Cited By ~ 32

Author(s):

Luca D. Passerini ◽

Zuzana Keckesova ◽

Greg J. Towers

Keyword(s):

Life Cycle ◽

Reverse Transcription ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Viral Life Cycle ◽

Human Cells ◽

Restriction Factors ◽

Retroviral Infection ◽

The Relationship ◽

Murine Leukemia

ABSTRACT The restriction factors Fv1 and TRIM5α provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5α-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5α for virus before reverse transcription. In human cells expressing Fv1b, N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5α by Fv1. Our findings suggest that, like TRIM5α, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5α when expressed in human cells.

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Hepatitis B Virus Capsids Have Diverse Structural Responses to Small-Molecule Ligands Bound to the Heteroaryldihydropyrimidine Pocket

Journal of Virology ◽

10.1128/jvi.03058-15 ◽

2016 ◽

Vol 90 (8) ◽

pp. 3994-4004 ◽

Cited By ~ 40

Author(s):

Balasubramanian Venkatakrishnan ◽

Sarah P. Katen ◽

Samson Francis ◽

Srinivas Chirapu ◽

M. G. Finn ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Drug Design ◽

Quaternary Structure ◽

Core Protein ◽

Viral Life Cycle ◽

Structural Basis ◽

Allosteric Modulators ◽

B Virus

ABSTRACTThough the hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-characterized activity is self-assembly into 240-monomer capsids. Small molecules that target core protein (core protein allosteric modulators [CpAMs]) represent a promising antiviral strategy. To better understand the structural basis of the CpAM mechanism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases protein-protein association more than 100-fold, and induces assembly of nonicosahedral macrostructures. In a preformed capsid, HAP18 is found at quasiequivalent subunit-subunit interfaces. In a detailed comparison to the two other extant CpAM structures, we find that the HAP18-capsid structure presents a paradox. Whereas the two other structures expanded the capsid diameter by up to 10 Å, HAP18 caused only minor changes in quaternary structure and actually decreased the capsid diameter by ∼3 Å. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design.IMPORTANCEHepatitis B virus core protein has multiple roles in the viral life cycle—assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions—making it an attractive antiviral target. Core protein allosteric modulators (CpAMs) are an experimental class of antivirals that bind core protein. The most recognized CpAM activity is that they accelerate core protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has multiple conformations. We compare structures of capsids cocrystallized with different CpAMs and find that they also affect quaternary structure in different ways. These results suggest that the capsid “breathes” and is trapped in different states by the drug and crystallization. Understanding that the capsid is a moving target will aid drug design and improve our understanding of HBV interaction with its environment.

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N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808319115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8829-8834 ◽

Cited By ~ 44

Author(s):

Hasan Imam ◽

Mohsin Khan ◽

Nandan S. Gokhale ◽

Alexa B. R. McIntyre ◽

Geon-Woo Kim ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcription ◽

Mutational Analysis ◽

Regulatory Function ◽

Messenger Rnas ◽

Stem Loop ◽

B Virus ◽

A Site

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m6A site in the 5′ epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

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Analysis of the Cytosolic Domains of the Hepatitis B Virus Envelope Proteins for Their Function in Viral Particle Assembly and Infectivity

Journal of Virology ◽

10.1128/jvi.00621-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 11935-11945 ◽

Cited By ~ 43

Author(s):

Matthieu Blanchet ◽

Camille Sureau

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Self Assembly ◽

Inner Core ◽

Envelope Proteins ◽

Endoplasmic Reticulum Membrane ◽

Virus Envelope ◽

Particle Assembly ◽

B Virus

ABSTRACT The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.

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