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 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 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 Duck Hepatitis B Virus Requires Cholesterol for Endosomal Escape during Virus Entry

2008 ◽  
Vol 82 (21) ◽  
pp. 10532-10542 ◽  
Author(s):  
Anneke Funk ◽  
Mouna Mhamdi ◽  
Heinz Hohenberg ◽  
Jörg Heeren ◽  
Rudolph Reimer ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Biological Membranes ◽  
Endosomal Escape ◽  
Cholesterol Depletion ◽  
Virus Infectivity ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Viral Particles ◽  
B Virus

ABSTRACT The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.

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.

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