TY - JOUR
T1 - An in vitro system for the enzymological analysis of avian hepatitis B virus replication and inhibition in core particles
AU - Urban, Sinisa
AU - Urban, Severin
AU - Tyrrell, D. Lorne
N1 - Funding Information:
This research was funded by a grant from Glaxo-Wellcome Canada. Sinisa Urban was partly supported by an External Research Scholarship from Trinity College, Cambridge. The anti-DHBV core antisera was a kind gift from Dr Jesse Summers, and the labeled 3TC-TP was synthesized by Dr John Wilson. We are grateful to our colleagues Karl P. Fischer and Bill Addison for many helpful discussions. The manuscript was significantly improved by the critical comments of Rosanna Baker.
PY - 2000/3
Y1 - 2000/3
N2 - A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition. Copyright (C) 2000 Elsevier Science B.V.
AB - A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition. Copyright (C) 2000 Elsevier Science B.V.
KW - Antiviral inhibition
KW - Duck hepatitis B virus
KW - Polymerase
KW - Replicating cores
KW - Replication
KW - Reverse transcriptase
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U2 - 10.1016/S0166-3542(00)00071-1
DO - 10.1016/S0166-3542(00)00071-1
M3 - Article
C2 - 10771082
AN - SCOPUS:0034164010
SN - 0166-3542
VL - 45
SP - 185
EP - 197
JO - Antiviral Research
JF - Antiviral Research
IS - 3
ER -