AMINO-ACIDS ESSENTIAL FOR RNASE-H ACTIVITY OF HEPADNAVIRUSES ARE ALSOREQUIRED FOR EFFICIENT ELONGATION OF MINUS-STRAND VIRAL-DNA

The hepadnavirus P gene contains amino acid sequences which share homo logy with all known RNases H. In this study, we made four mutants in w hich single amino acids of the duck hepatitis B virus (DHBV) RNase H r egion were altered, In two of them, amino acids at locations comprisin g the putative catalytic site were changed, while the remaining mutant s had alterations at amino acids conserved among hepadnaviruses. Trans fection of these mutant genomes into permissive cells resulted in synt hesis of several discrete viral nucleic acid species, ranging in appar ent sizes from approximately 500 to 3,000 bp, numbered I, II, III, TV, and V. While the locations of the species were similar in all mutants , the proportions of the species varied among the mutants, Analysis of the nucleic acid species revealed that they were hybrid molecules of RNA and minus-strand DNA, indicating that the RNase H activity was mis sing or greatly reduced in these mutants, Primer extension experiments showed that the mutant viruses initiated minus-strand viral DNA synth esis normally, The 3' termini of minus-strand DNA in species II, III, and TV were mapped just downstream of nucleotides 1659, 1220, and 721, respectively, Species V contained essentially full-length minus-stran d viral DNA, A parallel amino acid change in the putative catalytic si te of the HBV RNase H domain resulted in accumulation of low-molecular -weight hybrid molecules consisting of RNA and minus-strand DNA and si milar in size and pattern to those seen with DHBV. These studies demon strate experimentally the involvement of the C-terminal portion of the P gene in RNase H activity in both DHBV and human hepatitis B virus a nd indicate that the amino acids essential for RNase H activity of hep adnavirus P protein are also important for the efficient elongation of minus-strand viral DNA.