Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis.
The genome of all hepadnaviruses has an open reading frame called the P gene, which encodes a polypeptide of 90 to 97 kDa. The product or products of this P gene are involved in multiple functions of the viral life cycle. These functions include a priming activity which initiates minus-strand DNA synthesis, a polymerase activity which synthesizes DNA by using either RNA or DNA templates (reverse transcriptase), a nuclease activity which degrades the RNA strand of RNA-DNA hybrids (RNase H), and involvement in packaging the RNA pregenome into nucleocapsids. In a previous study, we found that a single point mutation at position 711 in the duck hepatitis B virus (DHBV) P gene product RNase H domain prevented viral RNA packaging. In the present experiments, we have mutated additional conserved amino acids in the DHBV RNase H domain and examined the ability of viral genomes containing these mutations to package RNA and replicate viral DNA. Charged and sulfur group amino acids adjacent to Cys-711 were mutated. None of these mutants was defective in either RNA packaging or viral replication. We also tested a number of mutations on the basis of common elements in the crystal structures of Escherichia coli and human immunodeficiency virus reverse transcriptase RNase H enzymes and on the basis of the similarities of their amino acid sequences to those of the RNase H domains of DHBV and HBV. Our results revealed that the entire beta 4 strand and amino acids Leu-712, Leu-697, and Val-719 in the putative hydrophobic cores of the beta 4, alpha A, and alpha B regions, respectively, are involved in pregenomic RNA encapsidation. This suggests that the basic structure of the RNase H domain in the DHBV P gene product is required for viral RNA packaging. We used the in vitro DHBV minus-strand DNA priming system developed by Wang and Seeger (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992) to test the effect of RNase H packaging mutations on P gene product enzymatic activity. While all packaging-defective mutants tested maintained DNA priming activity, levels were decreased 5- to 20-fold compared with that of the wild-type genome. This observation suggests that the hepadnavirus RNase H domain plays a role in optimizing priming of minus-strand DNA synthesis.
Chen
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Department of Medicine, Stanford University School of Medicine, California 94305.
Robinson
W S
WS
Marion
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PL
D000595:Amino Acid Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis*; D004563:Electrochemistry; D015611:Hepatitis B Virus, Duck / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D012323:RNA Processing, Post-Transcriptional; D012367:RNA, Viral / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins; D016914:Ribonuclease H