Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus
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The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.Phyllanthus
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Transcription
Viral transformation
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Woodchuck hepatitis virus
DNA virus
Hepatitis D virus
Hepatitis B
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To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti‐hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild‐type virus infection, A1896 variant infection and dual infection. Higher titre of anti‐HBe was found in patients with no virus replication and in patients coinfected with the wild‐type virus and A1896 variant, which suggest that anti‐HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site‐directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.
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An in vitro system for the production of hepatitis B virus (HBV) particles was established by the transient expression of transfected HBV DNA using a human hepatocellular carcinoma cell, HuH-7, as a recipient. The 3.6- and 2.2-kilobase transcripts observed were similar to those in virus-infected liver cells. Both transcripts revealed the microheterogeneity of their 5' ends. The formation of virus- related particles subsequent to the RNA transcription was demonstrated. The core particles observed in the cytoplasm and the virus particles secreted in the culture medium con- tained the replicative intermediates of IBV DNA and banded at densities of 1.35-1.36 g/cm3 and 1.22-1.24 g/cm3, respec- tively. Furthermore, the in vitro mutagenesis of the template HBV DNA demonstrated that the P gene as well as the C gene products were essential for the production of HBV particles. Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis. Furthermore, HBV is known to be a major cause of human liver cancer. HBV DNA has the following unique structural characteristics (1). Two linear DNA strands (plus and minus) form a circular molecule by annealing each cohesive end region. The minus strand is 3.2 kilobases (kb) in size with a protein linked to its 5' end. The plus strand usually shows various degrees of incompleteness. Similar HBV-like animal viruses have been discovered in wood- chucks (woodchuck hepatitis virus), ground squirrels (ground squirrel hepatitis virus, GSHV), and ducks (duck hepatitis B virus, DHBV). The replication cycle of the virus was proposed by Summers and Mason based on their observations of the in vivo replication of DHBV (2). How- ever, the restricted host range of HBV and lack of a cell culture system for virus production have been major prob- lems hindering from understanding of HBV replication and transcription at the molecular level. In this investigation, an in vitro system for the production of HBV particles was established by the transient expression of transfected HBV DNA, using a human hepatocellular carcinoma cell as a recipient. The RNA transcription of HBV DNA, similar to that in virus infection in vivo, was observed. Furthermore, mutagenesis of the template HBV DNA indi- cated virus production to be absolutely dependent on the template plasmid and the P gene as well as the C gene products to be essential for the synthesis of HBV DNA.
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THE duck hepatitis B virus as well as the human hepatitis B virus (HBV) belong to the hepadnaviruses. The viral DNA acts as a template for the synthesis, by DNA polymerase (DNAp). This enzyme is a DNA-dependent DNAp. It is a target enzyme of anti-hepatitis drugs too. Based on Shui Qin (SQ) anti-hepatitis B virus we used DHBV DNAp as a model in studying
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Hepatitis B
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ABSTRACT Hepadnaviruses selectively package capsids containing mature double-stranded DNA (dsDNA) genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing single-stranded DNA (ssDNA) in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA, with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the “capsid packaging signal” on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions than do those replicating DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis. IMPORTANCE Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that “capsid maturation,” and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication.
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Defective hepatitis B virus (HBV) particles, generated from singly spliced HBV RNA, have been detected in chronic carriers of HBV. The present study was designed to quantify the expression of defective HBV (dHBV) and wild-type HBV (wtHBV) genomes in the serum of patients with HBV infection and its relation to the severity of liver disease.HBV and dHBV loads were determined by quantitative polymerase chain reaction in the serum of 89 untreated HBV-infected patients (31 coinfected with human immunodeficiency virus [HIV] type 1) with liver disease of different stages. The ratio of dHBV DNA to total (wtHBV plus dHBV) HBV DNA (dHBV/HBV ratio) was used to express data independently of the level of viral replication.Despite a global correlation between dHBV and wtHBV load, the dHBV/HBV ratio ranged from 0.001% to 69%. The variation in dHBV/HBV ratio was independent of HIV coinfection, HBV genotype, and precore mutations. The mean dHBV/HBV ratio was higher in patients with severe liver necrosis and fibrosis.Our data indicate that an elevated dHBV/HBV ratio is associated with liver necroinflammation and fibrosis disease, suggesting a regulation of dHBV expression according to the severity of the liver disease. The dHBV/HBV ratio may help to better define liver disease stage during HBV infection.
Liver disease
Hepatitis B
Orthohepadnavirus
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Abstract To study the replicative efficiency and pathogenicity of hepatitis B virus precore variant (A1896), anti‐hepatitis B virus e antigen (HBe) titre was studied in naturally occurring wild‐type virus infection, A1896 variant infection and dual infection. Higher titre of anti‐HBe was found in patients with no virus replication and in patients coinfected with the wild‐type virus and A1896 variant, which suggest that anti‐HBe may either act as an inhibitor of virus replication or as selective pressure for the A1896 variant. Three site‐directed mutants were constructed in the duck hepatitis B virus (DHBV) precore region. A frame shift in the encapsidation signal region abolished replication of DHBV; mutation in the initiation codon of the precore and mutation to generate a termination codon at the distal region of the precore resulted in decreased replication in the duck model. More significant pathological changes were found in the liver tissues of ducks infected with the mutant which mimicked the HBV A1896 variant.
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