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Initiation of hepatitis delta virus genome replication.

ABSTRACT: The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (deltaAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant deltaAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input deltaAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of deltaAg-S. It is this second source of deltaAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for deltaAg-S with truncated or modified forms of the deltaAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of deltaAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

SUBMITTER: Dingle K

PROVIDER: S-EPMC110015 | biostudies-literature | 1998 Jun

REPOSITORIES: biostudies-literature

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Initiation of hepatitis delta virus genome replication.

Dingle K K Bichko V V Zuccola H H Hogle J J Taylor J J

Journal of virology 19980601 6

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (deltaAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant deltaAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or imm ...[more]

PMID: 9573243

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Project description:Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

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Initiation of hepatitis delta virus genome replication.

Publications

Initiation of hepatitis delta virus genome replication.

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