Initiation of Replication

In a natural infection, a receptor-mediated interaction of the virus with the host cell leads to the viral RNP reaching the nucleus and then initiating RNA-

directed RNA transcription. Such infections have also been initiated using cultured primary hepatocytes but never with established cell lines. To initiate HDV replication in cell lines, many different strategies have been used. Some are listed in Table 2.

Several important points need to be made about these systems: (1) S-HDAg has to be present at or soon after the transfection; (2) the in vitro RNAs and the in vivo DNA-directed transcripts are linear RNAs, not circular, as in the virions; (3) in some cases, even the total nucleic acid extracted from a cell undergoing replication, can be transfected into new cells and is sufficient to initiate genome replication; (4) for the method described in Sect. 3.1.3, a low level of replication is initiated by transfecting HDV RNA into cells continuously expressing only a low level of S-HDAg. When these cells are induced by addition of TET to express large amounts of S-HDAg, there can be a rapid burst of replication; presumably this is because the RNA templates are already circular species resident within the cells.

It has to be realized that in many transfection studies the HDV replication is initiated by nucleic acid species that are different from the circular genomic RNA present in the virions that initiate a natural infection. For some of these transfections the initial HDV RNA templates were greater than unit-length tandem multimers. It was considered that since each unit-length RNA, be it genomic or antigenomic, will contain a functional ribozyme, then initiation of replication might need an initial conversion of the RNA to unit-length linear and then to a circle, presuming that such circles might be the preferred template for RNA-directed transcription. While this maybe true, recent studies show that linear RNAs, even unit-length RNAs, do not necessarily have to

Table 2 Components used for transfections that can initiate HDV genome replication in cell lines

1. HDV virions (Bichko etal. 1994)

2. HDV RNP from virions (Bichko et al. 1994)

3. HDV cDNA in expression vectors (Kuo et al. 1989)

4. HDV RNA transcribed in vitro, into cells expressing S-HDAg (Glenn et al. 1990)

5. HDV RNA transcribed in vitro, pre-mixed with recombinant small S-HDAg (Dingle et al. 1998a)

6. HDV RNA transcribed in vitro, together with in vitro transcribed mRNA that can be translated into S-HDAg (Modahl and Lai 1998)

7. As in 4, but using total RNA extracted from cells in which replication was occurring (Gudima et al. 2004)

be converted to circles before they can be transcribed to initiate replication (Chang and Taylor 2002). This result has made us aware that even after the initiation a natural infection, some of the nascent HDV RNA species that are noncircular and possibly of unit length or greater, might actually function as templates for transcription. To assess this contribution, a competition assay between RNA circles and linear HDV RNAs was able to show that unit-length circles are about 15 times better at initiation than linear RNAs. Also, genomic and antigenomic RNAs are of equal efficiency in initiation (Gudima et al. 2004).

Many questions remain regarding the initiation of replication in a natural infection. Does S-HDAg function directly in this process and if so, how? What site(s) on the genomic RNA does the host polymerase recognize to facilitate such initiation? From what sites does the initiation actually take place? Are we correct in presuming that the 5'-end of the mRNA arose via site-specific primer-independent initiation? Later, when new antigenomic RNAs are produced, again what does the host polymerase bind to and where is transcription initiated from?

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