PREVIOUSLY UNSUSPECTED CIS-ACTING SEQUENCES FOR DNA-REPLICATION REVEALED BY CHARACTERIZATION OF A CHIMERIC HERON DUCK HEPATITIS-B VIRUS/

Heron hepatitis B virus (HHBV) is an avian hepadnavirus that is closel y related to duck hepatitis B virus (DHBV). To learn more about the me chanism of hepadnavirus replication, we characterized a clone of HHBV that contains a substitution of DHBV sequence from nucleotide coordina tes 403 to 1364. This clone, named HDE1, expresses a chimeric pregenom ic RNA, a chimeric polymerase (P) protein, and a core (C) protein with a one-amino-acid substitution at its carboxy terminus. We have shown that HDE1 is defective for minus-strand DNA synthesis, resulting in an overall reduction of viral DNA, HDE1 was also defective for plus-stra nd DNA synthesis, resulting in aberrant ratios of replication intermed iates, Genetic complementation assays indicated that HDE1 replication proteins, C and P, are functional for replication and wild-type HHBV p roteins do not rescue either defect, These findings indicate that the HDE1 substitution mutation acts primarily in cis, By restoring nucleot ides 403 to 902 to the HHBV sequence, we showed that cis-acting sequen ces for plus-strand DNA synthesis are located in the 5' half of the HD E1 chimeric region. These data indicate the presence of one or more fo rmerly unrecognized cis-acting sequences for DNA synthesis within the chimeric region (nucleotides 403 to 1364), These cis-acting sequences in the middle of the genome might interact directly or indirectly with known cis elements that are located near the ends of the genome. Our findings suggest that a specific higher-order template structure is in volved in the mechanism of hepadnavirus DNA replication.