Enzymology of RNADirected Transcription

Given that HDV genome replication involves RNA-directed RNA transcription andthatS-HDAgistoo smalltohavepolymeraseactivity, it hasbeenclear for some time that one or more host polymerases are required for HDV transcription. This being said, the characterization of such transcription leaves a lot to be desired.

Much evidence invokes the interpretation that replication involves the redirection of host DNA-directed RNA polymerase II, Pol II (Macnaughton et al. 2002; Modahl et al. 2000; Moraleda and Taylor 2001).

However, a complication is that it has been interpreted that a second polymerase, one that is more resistant to alpha-amanitin than Pol II, might be involved in the transcription of genomic RNA templates to produce antige-nomic RNAs (Macnaughton et al. 2002; Modahl et al. 2000). In the absence of convincing experimental support it has been interpreted that RNA polymerase I is involved. This in turn has been incorporated into a highly speculative and complex rolling-circle model in which genomic RNA templates are sometimes transcribed by Pol II and other times by Pol I (Macnaughton et al. 2002).

It is agreed that the transcription of antigenomic RNA templates into genomic RNA is sensitive to alpha-amanitin at levels consistent with the enzyme being Pol II. The accumulation of the HDV mRNA species is similarly sensitive. Furthermore, there is the circumstantial evidence for Pol II, in that accumulation of this mRNA is dependent upon poly(A) processing signals that in animal cells are only recognized by Pol II (Gudima et al. 2000; Hsieh and Taylor 1991; Nie et al. 2004).

One would hope that stronger evidence obtained via robust experimental systems might be available for this important transcription question. The problem is that to date, no one has been able to obtain a reproducible and competent system for in vitro transcription of HDV RNA templates. One early in vitro study cannot be reproduced (Fu and Taylor 1993). Other in vitro studies achieve what is predominantly 3'-end addition to HDV RNAs, a process that might not be of biological relevance (Beard et al. 1996; Filipovska and Konarska 2000; Gudima et al. 2000).

Why then has HDV transcription not been better characterized? The answer might be that no one has been able to use in vitro transcription reactions to achieve credible initiation of HDV RNA transcripts. In part, this may be because in vitro transcription will probably make use of ribonucleoprotein structures rather than naked HDV RNAs. Currently some progress is being made by the application of immunoaffinity procedures following disruption of cells undergoing a burst of HDV replication (Nie, Chang, Taylor, unpublished), just as others have done for other RNA viruses (Qanungo et al. 2004; Waris et al. 2004). Following such selections, both genomic and antigenomic unit-length HDV RNAs can be found bound to S-HDAg, and a fraction of these are also bound to RNA polymerase II (Nie, Chang, Taylor, unpublished). However, such complexes will have to be proven as competent for in vitro transcription and they will have to be carefully characterized for all the host proteins present.

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