scholarly journals Itinerary of Hepatitis B Viruses: Delineation of Restriction Points Critical for Infectious Entry

2004 ◽  
Vol 78 (15) ◽  
pp. 8289-8300 ◽  
Author(s):  
Anneke Funk ◽  
Mouna Mhamdi ◽  
Li Lin ◽  
Hans Will ◽  
Hüseyin Sirma
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
Viral Particles ◽  
B Virus ◽  
Number Of Binding Sites ◽  
Infectious Entry ◽  
First Time

ABSTRACT Little is known about cellular determinants essential for human hepatitis B virus infection. Using the duck hepatitis B virus as a model, we first established a sensitive binding assay for both virions and subviral particles and subsequently elucidated the characteristics of the early viral entry steps. The infection itinerary was found to initiate with the attachment of viral particles to a low number of binding sites on hepatocytes (about 104 per cell). Virus internalization was fully accomplished in less than 3 h but was then followed by a period of unprecedented length, about 14 h, until completion of nuclear import of the viral genome. Steps subsequent to virus entry depended on both intact microtubules and their dynamic turnover but not on actin cytoskeleton. Notably, cytoplasmic trafficking of viral particles and emergence of nuclear covalently closed circular DNA requires microtubules during entry only at and for specific time periods. Taken together, these data disclose for the first time a series of steps and their kinetics that are essential for the entry of hepatitis B viruses into hepatocytes and are different from those of any other virus reported so far.

scholarly journals Conditional Replication of Duck Hepatitis B Virus in Hepatoma Cells

2003 ◽  
Vol 77 (3) ◽  
pp. 1885-1893 ◽  
Author(s):  
Ju-Tao Guo ◽  
Melissa Pryce ◽  
Xingtai Wang ◽  
M. Inmaculada Barrasa ◽  
Jianming Hu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Biochemical Analysis ◽  
Duck Hepatitis B Virus ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
Viral Particles ◽  
B Virus ◽  
Host Cell Interactions ◽  
The Stability

ABSTRACT To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.

scholarly journals Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

Virology ◽  
2013 ◽  
Vol 446 (1-2) ◽  
pp. 357-364 ◽  
Author(s):  
Georget Y. Reaiche-Miller ◽  
Michael Thorpe ◽  
Huey Chi Low ◽  
Qiao Qiao ◽  
Catherine A. Scougall ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Duck Hepatitis B Virus ◽  
Circular Dna ◽  
Covalently Closed Circular Dna ◽  
Duck Hepatitis ◽  
B Virus

scholarly journals Cellular Receptor Traffic Is Essential for Productive Duck Hepatitis B Virus Infection

2000 ◽  
Vol 74 (5) ◽  
pp. 2203-2209 ◽  
Author(s):  
Klaus M. Breiner ◽  
Heinz Schaller
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Transmembrane Protein ◽  
Cellular Receptor ◽  
Receptor Function ◽  
Wild Type ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.

scholarly journals Small DNA Hairpin Negatively Regulates In Situ Priming during Duck Hepatitis B Virus Reverse Transcription

2002 ◽  
Vol 76 (3) ◽  
pp. 980-989 ◽  
Author(s):  
Jeffrey W. Habig ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Minus Strand ◽  
Dna Hairpin ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
B Virus ◽  
Pairing Potential

ABSTRACT There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to cis-acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5′ end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3′ end of the minus-strand DNA a poor template for initiation.

scholarly journals Mutations That Increase In Situ Priming Also Decrease Circularization for Duck Hepatitis B Virus

2001 ◽  
Vol 75 (14) ◽  
pp. 6492-6497 ◽  
Author(s):  
Daniel D. Loeb ◽  
Ru Tian
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Minus Strand ◽  
Complementary Sequence ◽  
Duck Hepatitis B Virus ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
B Virus ◽  
Template Switch

ABSTRACT The process of hepadnavirus reverse transcription involves two template switches during the synthesis of plus-strand DNA. The first involves translocation of the plus-strand primer from its site of generation, the 3′ end of minus-strand DNA, to the complementary sequence DR2, located near the 5′ end of the minus-strand DNA. Plus strands initiated from DR2 are extended to the 5′ end of the minus-strand DNA. At this point, the 3′ end of the minus strand becomes the template via the second template switch, a process called circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA. Although most virions contain relaxed circular DNA, some contain duplex linear DNA. Duplex linear genomes are synthesized when the plus-strand primer is used at the site of its generation, the 3′ end of the minus-strand template. This type of synthesis is called in situ priming. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within the 3′ end of the minus-strand DNA can lead to increased levels of in situ priming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-strand synthesis and the template switch that circularizes the genome.

scholarly journals Age-Related Differences in Amplification of Covalently Closed Circular DNA at Early Times after Duck Hepatitis B Virus Infection of Ducks

2005 ◽  
Vol 79 (15) ◽  
pp. 9896-9903 ◽  
Author(s):  
Yong-Yuan Zhang ◽  
Daniel P. Theele ◽  
Jesse Summers
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Duck Hepatitis B Virus ◽  
Circular Dna ◽  
Covalently Closed Circular Dna ◽  
Duck Hepatitis ◽  
Age Related ◽  
B Virus ◽  
Spread Of Infection

ABSTRACT Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses.

scholarly journals Formation of Hepatitis B Virus Covalently Closed Circular DNA: Removal of Genome-Linked Protein

2007 ◽  
Vol 81 (12) ◽  
pp. 6164-6174 ◽  
Author(s):  
Weifan Gao ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Envelope Protein ◽  
Cultured Cells ◽  
Protein S ◽  
Host Factors ◽  
Junction Sequence ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
B Virus

ABSTRACT Hepatitis B virus (HBV) contains a small, partially double-stranded, relaxed circular (RC) DNA genome. RC DNA needs to be converted to covalently closed circular (CCC) DNA, which serves as the template for all viral RNA transcription. As a first step toward understanding how CCC DNA is formed, we analyzed the viral and host factors that may be involved in CCC DNA formation, using transient and stable DNA transfections of HBV and the related avian hepadnavirus, duck hepatitis B virus (DHBV). Our results show that HBV CCC DNA formed in hepatoma cells was derived predominantly from RC DNA with a precise junction sequence. In contrast to that of DHBV, HBV CCC DNA formation in cultured cells was accompanied by the accumulation of a RC DNA species from which the covalently attached viral reverse transcriptase (RT) protein was removed (protein-free or PF-RC DNA). Furthermore, whereas envelope deficiency led to increased CCC DNA formation in DHBV, it resulted mainly in increased PF-RC, but not CCC, DNA in HBV, suggesting that the envelope protein(s) may negatively regulate a step in CCC DNA formation that precedes deproteination in both HBV and DHBV. Interestingly, PF-RC DNA, in contrast to RT-linked RC DNA, contained, almost exclusively, mature plus-strand DNA, suggesting that the RT protein was removed preferentially from mature RC DNA.

scholarly journals Analysis of Duck Hepatitis B Virus Reverse Transcription Indicates a Common Mechanism for the Two Template Switches during Plus-Strand DNA Synthesis

2002 ◽  
Vol 76 (6) ◽  
pp. 2763-2769 ◽  
Author(s):  
Michael B. Havert ◽  
Lin Ji ◽  
Daniel D. Loeb
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Synthesis ◽  
Minus Strand ◽  
Duck Hepatitis B Virus ◽  
Circular Dna ◽  
Duck Hepatitis ◽  
Donor And Acceptor ◽  
B Virus ◽  
Template Switches

ABSTRACT The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5′r and 3′r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5′ end, the middle, and the 3′ end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3′ of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism.

scholarly journals Carboxypeptidase D Is an Avian Hepatitis B Virus Receptor

1999 ◽  
Vol 73 (10) ◽  
pp. 8696-8702 ◽  
Author(s):  
Shuping Tong ◽  
Jisu Li ◽  
Jack R. Wands
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Virus Binding ◽  
Duck Hepatitis ◽  
Viral Particles ◽  
B Virus ◽  
Carboxypeptidase D ◽  
Immortalized Cell

ABSTRACT The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4°C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.

Sign in / Sign up

Export Citation Format

Share Document