HDAg sequence

Journal of General Virology (1993), 74, 24735477. Printed in Great Britain

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Characterization of RNA-binding domains of hepatitis delta antigen Francis P o i s s o n , ~ Philippe Roingeard, ~ Armelle Baillou, ~ Frederic D u b o i s , 1 Fabrizio Bonelli, 2 Raffaele A. C a l o g e r o 3 and M a i n G o u d e a u 1. 1Unitd de Virologie, D@artement de Microbiologie Mddicale et Moldculaire, U R A C N R S 1334, C H U Bretonneau, 2 Boulevard Tonnelld, 37044 Tours Cedex, France, 2 Research and Development S O R I N Biomedica S.p.A., Saluggia 13040 and 3 Department o f Genetics, General and Molecular Biology, University of Naples, Napoli 8100, Italy

Hepatitis delta antigen (HDAg), the only protein encoded by the hepatitis delta virus (HDV), binds specifically genomic and antigenomic strands of the HDV RNA. In a previous study, three recombinant HDAg subdomains were synthesized, covering residues 11 to 78, 79 to 163 and 164 to 212, and only the middle domain was shown to be responsible for the binding to HDV RNA. To investigate HDAg sequences involved in HDV RNA binding, we synthesized five peptides, 15 to 29 residues in length, and tested their ability to bind HDV RNA using a simple non-radioactive ELISA with

digoxigenin-labelled HDV genomic or antigenomic RNA probes. The specificity of interactions was demonstrated by comparison with control peptides and nonHDV RNA probes, and with an inhibition assay using recombinant HDAg. The HDAg-binding domain found within the middle region (79 to 163) of HDAg was more finely mapped: it is located between residues 79 and 107. In addition, another domain (residues 2 to 27) of HDAg was also found to bind specifically to HDV RNA. These two peptides share sequence similarities at residues 2 to 10 and 97 to 107 with other RNA-binding domains.

Hepatitis delta virus (HDV) is a viroid-like RNA agent which is dependent on the envelope of hepatitis B virus (HBV) acting as a helper virus (Rizzetto et al., 1980) for packaging into virions (Wang et al., 1991). The HDV genome is a circular ssRNA of approximately 1'7 kb (Wang et al., 1986; Makino et al., 1987) able to fold on itself by base-pairing to form an unbranched rod-like structure (Chen et al., 1986; Kos et al., 1986; Wang et al., 1986; Branch et al., 1990). The genome is replicated in the nucleus of infected cells by RNA-directed RNA synthesis, involving the synthesis of a complementary RNA (antigenome) which is also circular (Branch & Robertson, 1984; Chen et aI., 1986). Hepatitis delta antigen (HDAg) is the only known protein expressed by HDV. It is encoded by antigenomic RNA (Wang et al., 1986; Makino et al., 1987; Chang et aL, 1988 ; Kuo et al., 1988 ; Weiner et aL, 1988; Bergmann et al., 1989). HDAg is found in the nucleus of infected cells and is associated with genomic RNA in the mature virion. Two types of HDAg of 195 (S) and 214 (L) amino acids co-exist in infected cells (Bergmann & Gerin, 1986; Bonino et al., 1986; Pohl et al., 1987; Weiner et al., 1988). They are encoded by two RNA species: a single base change in the amber termination codon of the open reading frame of the S form leads to the synthesis of the L form which is 19 amino acids longer (Wang et al., 1986; Chao et al., 1990). The S form is essential for HDV

genome replication, whereas the L form acts as a dominant negative repressor of such replication (Macnaughton et aL, 1990a, 1991 ; Glenn & White, 1991 ; Xia et al., 1992; Chang et al., 1992). However, the L form is essential for the packaging of the genome into the HBV envelope (Chang et al., 1991 ; Taylor et al., 1991 ; Wang et al., 1991 ;Chen et al., 1992; Ryu et al., 1992). Previous studies have demonstrated that both the S and L forms of HDAg are able to bind either genomic or antigenomic HDV RNA (Chang et aL, 1988; Lin et al., 1990; Macnaughton et al., 1990b; Chao et al., 1991 ; Gowans et al., 1991; Lai et al., 1991; Taylor et al., 1991). A requirement for this specific binding is that HDV RNA forms a rod-like structure (Chao et al., 1991). Specific H D A g / H D V RNA binding is believed to be involved in several steps in the virus life cycle, including (i) nuclear transport of the genomic strand during the early phase of cellular HDV infection, (ii) regulation of the replication of both genomic and antigenomic strands by the S and L forms and (iii) packaging of the genomic strand into the HBV envelope. To analyse protein sequences involved in this binding, Lin et al. (1990) genetically expressed three recombinant HDAg subdomains covering residues 11 to 78, 79 to 163 and 164 to 212. Only the central domain (residues 79 to 163) was found to bind HDV RNA (Lin et al., 1990). To characterize further HDAg sequences involved in

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Short communication