Functions of Delta Antigen Methylation

Methylation of S-HDAg plays very important roles in HDV RNA replication. Methylated HDAg is likely to be essential for the initiation of replication of genomic RNA from the antigenomic strand. How does methylation of S-HDAg affect HDV replication?

Protein arginine methylation has been demonstrated to modulate transcription (Chevillard-Briet et al. 2002; Davie and Dent 2002), nucleic acid-binding affinity (Boulanger et al. 2005), protein-protein interaction (Bedford et al. 2000; Kwak et al. 2003), and nuclear targeting (Shen et al. 1998). The most likely role for methylation in HDAg function is that methylation regulates its subcellular distribution, as has been frequently observed for the methylation of other RNA-binding proteins, such as hnRNP A2 (Nichols et al. 2000) and RNA helicase A (Smith et al. 2004). Wild-type S-HDAg generally formed speckles in the nucleus, but the R13A mutant protein was localized predominantly in the cytoplasm. Treatment with methylation inhibitors also showed similar cytoplasmic localization of wild-type S-HDAg. Considering that HDV RNA replication was suggested to occur in the nuclear speckle structures by RNA polymerase II machinery (which is believed to be responsible for the replication of HDV genomic RNA from antigenomic RNA template) (Bichko and Taylor 1996; Macnaughton et al. 2002; Modahl et al. 2000; Moraleda and Taylor 2001), unmethylated HDAg might thus affect HDV replication by dislocating HDV from the antigenomic RNA template. This model is further supported by the observation that introduction of RNP complexes containing HDV antigenomic RNA and unmethylated HDAg could not target to the nucleus while RNP containing genomic RNA with unmethylated HDAg did. However, although arginine methylation of some RNA binding proteins has been reported to affect RNA binding, analysis of RNA binding of in vitro-methylated and unmethylated S-HDAg by UV cross-linking or gel mobility shift did not show significant differences (Li et al. 2004).

Methylation on arginine residues of transcriptional factors, such as DSIF or Spt5, were shown to inhibit association with RNA polymerase II and transcriptional activity (Kwak et al. 2003). HDAg has been demonstrated to interact with polymerase II and affect Pol II activity (Yamaguchi et al. 2001). Perhaps methylation of S-HDAg affects its interaction with the cellular transcription machinery responsible for HDV RNA replication. That the methylation of S-HDAg is required for the replication of genomic RNA from antigenomic RNA and for the formation of the speckled structures in the nucleus further suggests that methylation on arginine may modulate the interaction of S-HDAg and the RNA polymerase II transcription machinery.

Lysine methylation was also identified on S-HDAg, but the effects have not yet been pursued. HIV Tat and histone 3 also undergo methylation and acetylation on lysine, and thus may offer some clues. Arginine methylation diminishes the transactivation capacity of Tat, in contrast to the positive effect of lysine acetylation on Tat activity (Boulanger et al. 2005). Meanwhile, lysine acetylation of some proteins at a site that also serves as a substrate for another modification (such as methylation on lysine 9 of histone 3 and sumoylation on lysine 539 of sp3) blocked such modification. If it is the case for S-HDAg, the balance of methylation and acetylation on lysine residues will offer more delicate control of the HDV life cycle.

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