Host RNA Polymerase Requirements for Transcription of the Human Hepatitis Delta Virus Genome

Abstract

Replication of the genome of hepatitis delta virus (HDV) requires RNA-directed RNA synthesis using a host polymerase(s). This manuscript reviews the relevant published evidence. It also provides two new studies, both of which made use of transiently transfected Huh7 cells undergoing HDV RNA-directed RNA synthesis. For the first study, RNA transcription inhibitors were added to the transfected cells for periods of 1 to 2 days, after which assays of the effects on the accumulation of processed unit-length genomic HDV RNA were performed. For the second study, nuclei were isolated at 6 days after transfection, and then in vitro runoff transcription was used to assay the effects of RNA transcription inhibitors. Overall, the data support the interpretation that HDV transcription does not require host polymerase I or III (pol I or III) but at least primarily involves an enzyme resembling pol II.Plant viroids and the human hepatitis delta virus (HDV) have much in common (47). One of the more intriguing similarities is that they both replicate via RNA-directed RNA synthesis using a host RNA polymerase (44). Since there are no known DNA intermediates (5) the most likely explanation is that these agents with their single-stranded RNA genomes are able to redirect a host polymerase that normally uses DNA templates to use RNA as a template. An alternative explanation is that the host cell contains an RNA-directed RNA polymerase that can carry out the transcription. Actually, such a polymerase was first found and purified from plants (40). cDNA cloning of this polymerase (41) revealed that all plants and some animals contain at least one copy of a related gene. However, at this time, no such gene has been detected in mammals, which can be hosts for HDV.Several different suggestions have surfaced as to which host polymerases are involved. The plant viroids are frequently divided into two families. For one of these families there are data that transcription occurs in association with chloroplasts, using a nucleus-encoded polymerase (34). For the second viroid family, the available evidence implicates the host RNA polymerase II (pol II) (42,49). Similarly, with HDV there are many claims for pol II as the relevant enzyme (1, 12, 13, 26), although a recent paper has suggested the possibility that more than one host polymerase may be involved (31).For purposes of evaluation, the evidence for the polymerase(s) involved in HDV transcription may be divided into four different types: (i) circumstantial, (ii) in vivo, (iii) in vitro, and (iv) a combination of in vivo and in vitro.(i) Consider first the circumstantial evidence. During HDV genome replication the following three RNA species are detected (5): (a) the 1,679-nucleotide (nt) circular RNA genome; (b) its exact complement, the so-called antigenome; and (c) relatively low amounts of a less-than-unit-length antigenomic RNA that contains the open reading frame for the small delta protein, a 195-amino-acid species that is essential for HDV replication. This third RNA is thought to be...

Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs

“…Almost all the available evidence indicates that host pol II is the enzyme involved in HDV RNA-directed transcription (Modahl et al 2000;Moraleda and Taylor 2001;Macnaughton et al 2002;Taylor 2003). Therefore, the reconstitution results presented in this study suggest that RNA polymerase II is able to make correct intermolecular template-switching during RNA-directed RNA synthesis.…”

“…Much evidence supports the interpretation that the HDV genome is replicated by the host RNA polymerase II, an enzyme that is normally involved in DNA-directed host RNA synthesis (Modahl et al 2000;Moraleda and Taylor 2001;Macnaughton et al 2002;Taylor 2003). Furthermore, when HDV replication is initiated from linear RNA templates, this host polymerase achieves intramolecular template-switching Gudima et al 2004).…”

“…RNA 12 was an RNA of the same size and sequence as the mRNA known to arise during normal HDV genome replication (Gudima et al 2000). Moreover, this partner was transcribed so as to be like the natural mRNA, with a 5Ј-cap and a 3Ј-poly(A) sequence, and has been previously shown to be translated in vivo to yield the delta protein required to support HDV genome replication (Moraleda and Taylor 2001). This experiment was a positive control for the ability of a mRNA to interact with an RNA, such as RNA 11, and achieve reconstitution.…”

“…The challenge now is to determine to what extent such studies based on transcription from DNA templates can be extrapolated to the same enzyme when it acts in vivo to not only copy templates that are RNA but also to carry out template-switching that can be both intraand intermolecular. A current limitation is that, so far, no one has achieved a robust system for HDV RNA-directed transcription in vitro (Beard et al 1996;Filipovska and Konarska 2000;Gudima et al 2000;Moraleda and Taylor 2001;Yamaguchi et al 2001). As an alternative approach, HDV RNA-directed transcription is currently being studied using immuno-precipitation of transcription complexes that have been formed in vivo (X. Nie, J. Chang, and J.M.…”

Searching for nuclear export elements in hepatitis D virus RNA

“…The monomeric antigenomes serve as templates for a second round of replication, by a similar mechanism, thus resulting in the synthesis of monomeric genomic RNA molecules. The HDV genome contains a single ORF that codes for a 24 kDa protein, the so-called small delta antigen (S-HDAg) [9] . As a consequence of an editing mechanism that converts an amber stop codon UAG into a tryptophan codon UGG in the antigenome, the ORF is extended by 19 additional aminoacids [10] .…”