Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.Hepatitis delta virus (HDV) causes chronic and, occasionally, fulminant hepatitis in humans (16). HDV is a satellite virus which requires hepatitis B virus (HBV) to supply envelope proteins for virus assembly and production (46). It contains a circular RNA genome of 1.7 kb which replicates through a double-rolling-circle mechanism in the nucleus (33). The viral genomic RNA (G-RNA) is first replicated into the full-length antigenomic RNA (AG-RNA) and is also transcribed into a 0.8-kb mRNA, which encodes the only HDV protein, hepatitis delta antigen (HDAg). The AG-RNA, in turn, is replicated into G-RNA by another round of rolling-circle replication. The production of HDAg, which is intimately involved in HDV RNA replication, is a unique feature distinguishing HDV from plant viroids, which do not encode any protein. HDAg consists of two species, the small delta antigen (S-HDAg) (195 amino acids [aa]; 24 kDa) and the large delta antigen (L-HDAg) (214 aa; 27 kDa), which play different roles in HDV replication. S-HDAg is an essential activator for HDV RNA replication (25). In contrast, L-HDAg inhibits certain stages of HDV RNA replication but is required for virion assembly (5, 7, 28, 32). HDAg has been shown to be modified posttranslationally by phosphorylation (6, 9, 40, 42), acetylation (41), methylation (29), and in the case of L-HDAg, isoprenylation (14). Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation are three major modifications of S-HDAg and are important for the functions of S-HDAg in HDV RNA replication (29, 40, 41, 48). Isoprenylation on Cys-211 of L-HDAg is required for virus assembly (14).SUMO (small ubiquitin-related modifier) has been identified as a reversible posttranslational protein modifier (34, 35). The human genome encodes four SUMO proteins: SUMO1 to SUMO4 (13, 17). Among them, SUMO1 to SUMO3 are ubiquitously expressed, whereas SUMO4 is expressed mainly in the kidneys, lymph nodes, and spleen (17). Sumoylation is carried out by an E1 activating enzyme (the heterodimer Uba2-Aos1), an E2 conjugating enzyme (Ubc9), and one of several SUMO E3 ligases (38). Although SUMO E1 and E2 are sufficient to modify most substrates in vitro, in vivo sumoylation is usually facilitated by SUMO E3 for substrate selection (13, 24, 44). SUMO modification is a highly dynamic process that can be reversed rapidly by the action of SUMO-specific proteases, which are also involved in the maturation of newly synthesized SUMO proteins (18, 43). Although sumoylated proteins have been found throughout the cell, most of the known SUMO targets are cellular and viral proteins that function in the nucleus (19, 56). The functional outcomes of sumoylation are extremely diverse, including changes in intracellular localization, protein-protein interaction, protein-DNA interaction, and stability and activity of modified proteins (2, 11, 52, 56).Here we show that HDV S-HDAg is posttranslationally modified by the SUMO pathway both in vivo and in vitro. Using a genetic fusion chimera to mimic sumoylated S-HDAg, we found that SUMO1 conjugation of S-HDAg selectively enhances HDV G-RNA and mRNA synthesis but not AG-RNA synthesis. This result adds to the growing list of different metabolic requirements between HDV G-RNA/mRNA synthesis and AG-RNA synthesis and supports the hypothesis that the cellular machinery involved in the synthesis of HDV AG-RNA is different from that for G-RNA synthesis and mRNA transcription.