An in vitro system for the enzymological analysis of avian hepatitis B virus replication and inhibition in core particles

• Published in: Antiviral research Vol. 45; no. 3; pp. 185 - 197
• Main Authors: Urban, Sinisa, Urban, Severin, Tyrrell, D.Lorne
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2000; Elsevier
• Subjects: Animals; Antiviral inhibition; Biological and medical sciences; Chromatography, Agarose; Chromatography, Gel; Detergents; Duck hepatitis B virus; Ducks; Fundamental and applied biological sciences. Psychology; Hepatitis B virus; Hepatitis B Virus, Duck - chemistry; Hepatitis B Virus, Duck - enzymology; Hepatitis B Virus, Duck - isolation & purification; Microbiology; Polymerase; Replicating cores; Replication; Reverse transcriptase; RNA-Directed DNA Polymerase - chemistry; RNA-Directed DNA Polymerase - metabolism; Techniques used in virology; Time Factors; Viral Core Proteins - chemistry; Viral Core Proteins - isolation & purification; Virology; Virus Replication; Duck hepatitis B virus; Polymerase; Antiviral inhibition; Replicating cores; Replication; Reverse transcriptase; Purification; RNA-directed DNA polymerase; Enzyme; Transferases; Core particle; In vitro; Virus; Nucleotidyltransferases; Analysis method; Enzymatic activity; Avihepadnavirus; Hepadnaviridae; Isolation
• ISSN: 0166-3542; 1872-9096
• DOI: 10.1016/S0166-3542(00)00071-1

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.