Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins

Schlicht, H J; Radziwill, Gerald; Schaller, Heinz

doi:10.1016/0092-8674(89)90986-0

Cited by 129 publications

(82 citation statements)

References 32 publications

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“…Because S gene replacement destroys the surface protein and polymerase ORFs, it was necessary for the generation of recombinant virus that the corresponding gene products be trans-complemented by cotransfection (24,38) with the respective encapsidationdeficient helper construct (38,39). The result was the production of enveloped recombinant HBV (rHBV) at titers between 10 8 and 10 9 vp͞ml and recombinant DHBV (rDHBV) at titers between 3 ϫ 10 7 and 2.5 ϫ 10 8 vp͞ml in different experiments, comparable to the production of wild-type HBV and DHBV obtained by transfection (19).…”

Section: Resultsmentioning

confidence: 99%

Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection

Protzer

Nassal

Chiang

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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106

127

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Hepatitis B viruses specifically target the liver, where they efficiently infect quiescent hepatocytes. Here we show that human and avian hepatitis B viruses can be converted into vectors for liver-directed gene transfer. These vectors allow hepatocyte-specific expression of a green f luorescent protein in vitro and in vivo. Moreover, when used to transduce a type I interferon gene, expression of interferon efficiently suppresses wild-type virus replication in the duck model of hepatitis B virus infection. These data suggest local cytokine production after hepatitis-B-virus-mediated gene transfer as a promising concept for the treatment of acquired liver diseases, including chronic hepatitis B.

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Section: Resultsmentioning

confidence: 99%

Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection

Protzer

Nassal

Chiang

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

127

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“…Cloning and Transfection-Mutant S245A and mutant S245D were generated by cloning the 391-bp EcoRI/AvrII fragment of the corresponding core protein expression vectors described by Yu and Summers (11,16) into the overlength DHBV genome plasmid pOL16 (18). Mutant S245N was generated by cloning a PCR amplification product, in which the serine 245-coding triplet TCC was substituted by the asparaginecoding triplet AAC.…”

Section: Methodsmentioning

confidence: 99%

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

Köck

Kann

Pütz

et al. 2003

Journal of Biological Chemistry

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Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acidsupported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wildtype and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.Hepadnaviruses are a group of small enveloped DNA viruses with a narrow host range and a relative tropism for the liver. Hepatitis B virus (HBV), 1 the prototypic member of the hepadnavirus family, is a major cause of liver disease worldwide, ranging from acute and chronic hepatitis to liver cirrhosis and hepatocellular carcinoma (1, 2). Other members of the family include the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus (WHV) (3, 4). Similar to other viruses the hepadnaviral genome is protected by a nucleocapsid, which is formed by icosahedrally arranged core proteins. The C-terminal domain of the core protein is rich in basic residues and bears three conserved serine phosphorylation sites (5, 6). While dispensable for capsid assembly, this region is required for packaging of pregenomic RNA (7-11). Inside the capsid the viral polymerase converts pregenomic RNA into minus-strand DNA, which in turn is completed to a double-stranded, relaxed circular (rc) DNA molecule (12). Newly assembled, mature hepadnaviral capsids containing rcDNA have two possible fates: they can either be enveloped by surface proteins and enter the secretory pathway or be redirected to the nucleus where repair of the rcDNA molecule results in the formation of covalently closed circular (ccc) DNA, the template for viral RNA synthesis. Nuclear translocation of viral rcDNA is also mediated by incoming capsids in newly infected cells. However, the precise mechanisms regulating capsid trafficking and uncoating in both settings are unknown.Earlier studies in the DHBV model have shown that capsids from infected liv...

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“…Trans-complementation of the C and P genes of DHBV, recently reported by two independent groups (Chang et al, 1989;Schlicht et al, 1989), has led us to evaluate the capacity of DNA polymerases of heterologous hepadnaviruses to replicate a P gene defective mutant of HBV DNA in an in vitro transient expression system. HBV DNA clone (pPYW310; 3182 bp, subtype ayw), was isolated from serum of a Papua New Guinean blood donor, provided by Dr D. G. Woodfield of the New Zealand Red Cross Blood Centre.…”

Section: Trans-complementation Of the C Gene Of Human And The P Gene mentioning

confidence: 99%

“…The pol gene of retroviruses is expressed as a nucleocapsid-polymerase (gag-pol) fusion protein by ribosomal frameshifting during the translation of overlapping genes (Jacks & Varmus, 1985). The P gene of hepadnaviruses, in contrast, is expressed independently of the C gene by an internal initiation (Chang et al, 1989;Schlicht et al, 1989). As a consequence trans-complementation is achieved by a C gene defective mutant and a P gene defective mutant, for both HBV (Yaginuma et aL, 1987) and DHBV (Chang et al, 1989;Schlicht et al, 1989).…”

Section: Trans-complementation Of the C Gene Of Human And The P Gene mentioning

confidence: 99%

“…The P gene of hepadnaviruses, in contrast, is expressed independently of the C gene by an internal initiation (Chang et al, 1989;Schlicht et al, 1989). As a consequence trans-complementation is achieved by a C gene defective mutant and a P gene defective mutant, for both HBV (Yaginuma et aL, 1987) and DHBV (Chang et al, 1989;Schlicht et al, 1989). We initiating the synthesis of both plus-and minus-strand DNAs (Seeger et al, 1986;Lien et al, 1987;Will et al, 1987).…”

Section: Trans-complementation Of the C Gene Of Human And The P Gene mentioning

confidence: 99%

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Trans-complementation of the C gene of human and the P gene of woodchuck hepadnaviruses

Okamoto

Omi

Wang

et al. 1990

Journal of General Virology

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A 5 bp insertion was introduced into the BstEII site at nucleotide 2815 in DNA of hepatitis B virus (HBV) and a mutant HBV genome was produced, which coded for envelope and core proteins, but not for DNA polymerase, due to a frameshift. Cultured hepatoma ceils (HepG2) were simultaneously transfected with a plasmid harbouring a tandem dimer of the mutant HBV DNA and another plasmid harbouring a tandem dimer of DNA of woodchuck hepatitis virus or duck hepatitis B virus. The replication of mutant HBV DNA, incapable of encoding DNA polymerase, was accomplished by cotransfecting woodchuck hepatitis virus DNA, but not by duck hepatitis B virus DNA. These results indicated a trans-complementation of the C and P genes in mammalian hepadnaviruses beyond a species barrier.

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Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins

Cited by 129 publications

References 32 publications

Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection

Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

Trans-complementation of the C gene of human and the P gene of woodchuck hepadnaviruses

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