Effects of Excessive Editing at the AmberW Site

Overexpression of ADAR1 by cotransfection of ADAR1 expression constructs resulted in increased editing at the amber/W site, and increased production of L-HDAg (Jayan and Casey 2002a; Sato et al. 2004). Concomitantly, levels of HDV RNA synthesis were strongly inhibited. Another approach gave similar results: increasing the base-pairing 3' of the amber/W site led to higher levels of editing and L-HDAg synthesis, and dramatic inhibition of viral RNA synthesis (Jayan and Casey 2005; Sato et al. 2004). In all cases, overproduction of L-HDAg accounted for a significant fraction of the inhibition. The sensitivity of replication to editing (via L-HDAg) is remarkable; in one study mutations that increased editing by approximately threefold led to a 50-fold decrease in RNA replication (Jayan and Casey 2005).

It might be expected that inhibition of HDV RNA synthesis due to increased editing and subsequent L-HDAg overproduction would automatically inhibit viral particle production (because of decreased viral RNA levels within the cell). Indeed, inhibition ofvirus production was observed 6-12 days post-transfection with a site-directed mutant that exhibited increased editing (Jayan and Casey 2005). However, there was no inhibition of virus production before day 6, even though intracellular RNA levels were significantly decreased. One explanation of these results is that intracellular viral RNA is not normally the limiting factor for particle production. Virus secretion was closely correlated with levels of L-HDAg, consistent with the interpretation that L-HDAg is the limiting factor for virus particle production.

ADAR1 has several isoforms, one of which is induced by interferon. Although siRNA knockdown studies have shown that the shorter form of ADAR1, which is not induced by interferon, is primarily responsible for editing at the amber/W site (Wong and Lazinski 2002), treatment of Huh-7 cells with interferon has been shown to increase ADAR1 p150 expression and increase editing (Hartwig et al. 2004). Levels of HDV RNA were not analyzed in this study; based on the inhibition of replication associated with modest increases in editing due to ADAR co-transfection or editing site mutations (Jayan and Casey 2002a, 2005, Sato et al. 2004), it might be expected that HDV RNA levels would decrease. Conversely, previous studies indicated that interferon treatment of cultured cells did not affect HDV RNA replication (Ilan et al. 1992; McNair et al. 1994); however, the effects of interferon treatment on ADAR1 and amber/W editing were not assessed. Analysis of the effects of interferon on both editing and replication in the same study is required to clarify this issue.

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