Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus.
We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.
Roychoudhury
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Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104-6059.
Faruqi
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AF
Shih
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D001483:Base Sequence; D015152:Blotting, Northern; D002213:Capsid / Q000378:metabolism; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006515:Hepatitis B virus / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation; D014779:Virus Replication
D004279:DNA, Viral; D009841:Oligonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H