Replication of Genomic vs Antigenomic RNA Differences and Similarities

RNA replication strategies of all single-stranded RNA viruses incorporate regulatory mechanisms to control the relative amounts and, in many cases, the temporal synthesis of genomic and antigenomic RNA species. Since genomic and antigenomic HDV RNA are also synthesized in significantly different amounts, there must also be regulatory mechanisms operating during HDV replication. How this is achieved with just one virus-encoded protein is not clear. Certainly, both genomic and antigenomic RNA strands are synthe sized continuously throughout the replication cycle. Moreover, genomic and antigenomic RNA intermediates of similar length are detected (dimer and longer multimers) and the ratios of circular to linear RNA molecules in both strands are also similar (Macnaughton and Lai 2002a), indicating that the both strands are made by an analogous rolling circle mechanism. There are, however, some different metabolic requirements for the synthesis of the two strands (Table 1). Genomic RNA synthesis (from the antigenomic template) is inhibited by very low concentrations of a-amanitin (1 ^g/ml), whereas the antigenomic RNA synthesis (from the genomic template) is resistant to all concentrations tested (up to 100 ^g/ml; Macnaughton et al. 2002). In contrast, transcription of the antigenomic-sense 0.8-kb HDAg mRNA is as sensitive to a-amanitin as genomic RNA synthesis (Modahl et al. 2000). Genomic RNA synthesis also requires some specific S-HDAg post-translational modifications (acetylation, phosphorylation and methylation), whereas antigenomic RNA synthesis can be mediated by an unmodified S-HDAg. For example: phosphorylation-defective mutant (S177A) and methylation-defective mutant (R13A) of HDAg can promote antigenomic RNA synthesis, but not the genomic RNA synthesis (Mu et al. 2001; Li et al. 2004); acetylation-defective mutant K72R (Mu et al. 2004) causes a dramatic reduction in genomic RNA accumulation while having no effect on antigenomic accumulation (Table 1). Genomic RNA synthesis is inhibited by early expression of L-HDAg, whereas the antigenomic RNA synthesis is not (Modahl and Lai 2000). Genomic RNA is rapidly exported to the cytoplasm after its synthesis and processing, whereas the antigenomic RNA is retained in the nucleus (Macnaughton and Lai 2002a). Furthermore, several site-specific mutations of HDV RNA sequence affect the synthesis of one RNA strand but not the other (Wang et al. 1997). Finally, ge-nomic and antigenomic HDV RNA have different distribution patterns within the nucleus (Bell et al. 2000). Taken together, these results indicate that not only is the synthesis of genomic and antigenomic HDV RNA differentially regulated, but also that these two RNA strands are likely associated with different transcription machineries.

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