Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors.
Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg(2+) and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Juan Antonio
JA
Villa
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Daniel P
DP
Pike
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Kunjan B
KB
Patel
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Elena
E
Lomonosova
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA; Department of Gastroenterology, The Second Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, China.
Gaofeng
G
Lu
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Roz
R
Abdulqader
Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO 63104, USA. Electronic address: tavisje@slu.edu.
John E
JE
Tavis
D000998:Antiviral Agents / Q000494:pharmacology*; D055808:Drug Discovery; D015870:Gene Expression; D006515:Hepatitis B virus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D055503:Protein Multimerization; D059367:RNA Cleavage; D012367:RNA, Viral; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D000998:Antiviral Agents; D012367:RNA, Viral; D011994:Recombinant Proteins; D016914:Ribonuclease H
DNA virus; Endoribonuclease; Hepatitis B virus; RNA processing; Ribonuclease