Template Switching Reconstitution and Recombination

From studies of HDV replication as initiated by the transfection of linear HDV RNAs, it is clear that template-switching can occur during transcription of the transfected RNA. The best evidence for this is that when cells are transfected with linear RNAs that are one or two nucleotides less than unit-length, replication can be initiated but there are specific deletions and even nontemplated additions, on the RNAs that replicate (Chang and Taylor 2002).

With this knowledge that template-switching can occur, experiments were undertaken in which the transfected RNA was replaced by two RNAs, each of which was significantly less than unit-length, but which together provided representation of the whole genome. Following transfection with such RNAs, genome replication was detected, consistent with reconstitution of the HDV genome (Gudima et al. 2005). Such reconstitution was only achieved when the two RNA templates were pre-associated before the transfection. In fact, all of the available data concerning HDV template-switching is consistent with the role of the rod-like folding as a facilitator. That is, the inter-molecular association achieved prior to transcription depends on utilization of base-pairings normally considered to be part of the intra-molecular rod-like

Table 4 Some key steps in HDV RNA-directed RNA transcription during a natural infection

1. Following virus attachment and entry the genomic RNA, as a ribonucleoprotein (RNP) complex with S-HDAg, is transported to the nucleus

2. This RNP is able to redirect either an inactive Pol II complex or one already active in DNA-directed RNA transcription

3. One of the sites for initiation of transcription on the genomic RNA corresponds to position 1630, the 5'-end of the mRNA

4. Following initiation, elongation using the circular RNA template, can produce antigenomic transcripts that are greater than unit length

5. Such nascent antigenomic RNA transcripts can be processed either to mRNA or to unit-length circular antigenomic RNA, both of which are relatively much more stable than the nascent transcript

6. The mRNA species are transported to the cytoplasm and the translation product, new S-HDAg, returns to the nucleus to support more RNA-directed RNA transcription. It may also alter the balance of processing in step 5

7. Transcription of new antigenomic RNA templates occurs, with the nascent transcripts being processed to form new unit-length genomic RNAs

8. ADAR editing can occur on all nascent genomic and antigenomic RNAs, and/or on processed unit-length RNAs. Essential to the life cycle is that some nascent antigenomic RNA and/or processed unit-length antigenomic RNAs, become a target for specific ADAR-editing at position 1012, leading to the production and translation of mRNAs encoding L-HDAg

9. After editing and maybe other sequence changes, translation produces altered forms of HDAg, especially of L-HDAg, that fail to support or even act as inhibitors of further RNA-directed transcription. In addition, L-HDAg, after isoprenylation, can complex with genomic RNA that in turn can interact with the envelope proteins of the helper virus HBV, if present, to achieve assembly and release of new virus particles folding. Subsequently, it is considered that these new base-pairings within the RNA hybrid template force pauses in transcription at locations which allow template-switching to occur and achieve reconstitution of replication competent HDV RNA.

It might be argued that the ab ove examples of template-switching, although achieved within a cell rather than in vitro, are more relevant to the question of what an RNA polymerase can do, rather than to how an HDV genome is normally replicated. However, such studies are relevant to the question of whether there can be recombination between HDV RNA genomes.

Some data from examination of patients infected with two different HDV genotypes have been interpreted as evidence for inter-molecular recombina tion (Wang and Chao 2005; Wu et al. 1999). Also, it has been asserted that recombination can be achieved in cells transfected with two different HDV genotypes (Wang and Chao 2005). However, these data are not yet convincing and it is known that other attempts involving transfected RNAs have proven negative (Gudima et al. 2005).

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