PMID- 7687299
OWN - NLM
STAT- MEDLINE
DCOM- 19930816
LR  - 20200724
IS  - 0022-538X (Print)
IS  - 1098-5514 (Electronic)
IS  - 0022-538X (Linking)
VI  - 67
IP  - 8
DP  - 1993 Aug
TI  - Recombinant human hepatitis B virus reverse transcriptase is active in the 
      absence of the nucleocapsid or the viral replication origin, DR1.
PG  - 4513-20
AB  - The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed 
      from the viral pregenome RNA template by a virally encoded reverse transcriptase 
      enzyme (RT) that possesses both priming and elongation activities. Prior efforts 
      have failed to express an active form of HBV RT outside the nucleocapsid in 
      animal cells or to release it from viral nucleocapsids, thus restricting the 
      characterization of this important enzyme. Here, we have engineered 
      epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a 
      synthetic RT mRNA which does not include the viral capsid protein or the known 
      initiation site for viral DNA synthesis, DR1. We demonstrate the production of an 
      immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT 
      assay, that oocyte lysates containing this protein possess an activity that (i) 
      catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction 
      by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT 
      inhibitors actinomycin D and phosphonoformate. Experiments with the chain 
      terminator ddATP suggest that a significant amount of chain elongation occurs in 
      our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of 
      RT reaction products with sizes ranging from about 100 bases to far larger than 
      that of the input RT mRNA. These products appear to contain covalently bound 
      protein, consistent with the notion that the RT protein may have primed their 
      synthesis. We conclude that HBV RT activity can be uncoupled from both the 
      nucleocapsid and the replication origin, DR1. Our results raise the possibility 
      that unless HBV employs novel mechanisms to regulate its constitutively active 
      RT, cellular RNAs may be reverse transcribed during HBV infection, with potential 
      implications for the development of HBV-related liver cancer. The use of the 
      oocyte system should facilitate studies of HBV RT, including the development of 
      HBV RT inhibitors for antiviral therapy.
FAU - Seifer, M
AU  - Seifer M
AD  - Hormone Research Institute, University of California, San Francisco, California 
      94143-0534.
FAU - Standring, D N
AU  - Standring DN
LA  - eng
GR  - AI25056/AI/NIAID NIH HHS/United States
GR  - DK-26743/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Virol
JT  - Journal of virology
JID - 0113724
RN  - 0 (DNA, Viral)
RN  - 0 (Epitopes)
RN  - 0 (RNA, Messenger)
RN  - 0 (Recombinant Proteins)
RN  - EC 2.7.7.49 (RNA-Directed DNA Polymerase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Capsid/*metabolism
MH  - *DNA Replication
MH  - DNA, Viral/*biosynthesis/isolation & purification
MH  - Epitopes/analysis
MH  - Female
MH  - *Genome, Viral
MH  - Hepatitis B virus/*enzymology/genetics/physiology
MH  - Humans
MH  - Molecular Sequence Data
MH  - Oocytes/metabolism
MH  - Protein Biosynthesis
MH  - RNA, Messenger/metabolism
MH  - RNA-Directed DNA Polymerase/*metabolism
MH  - Recombinant Proteins/metabolism
MH  - Transcription, Genetic
MH  - Virus Replication
MH  - Xenopus
PMC - PMC237835
EDAT- 1993/08/01 00:00
MHDA- 1993/08/01 00:01
PMCR- 1993/08/01
CRDT- 1993/08/01 00:00
PHST- 1993/08/01 00:00 [pubmed]
PHST- 1993/08/01 00:01 [medline]
PHST- 1993/08/01 00:00 [entrez]
PHST- 1993/08/01 00:00 [pmc-release]
AID - 10.1128/JVI.67.8.4513-4520.1993 [doi]
PST - ppublish
SO  - J Virol. 1993 Aug;67(8):4513-20. doi: 10.1128/JVI.67.8.4513-4520.1993.