Initiation of Hepatitis Delta Virus Genome Replication

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (δAg-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 δAg-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 δAg-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 δAg-S. It is this second source of δAg-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 δAg-S with truncated or modified forms of the δAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of δAg-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.