Structural Organization of the Hepatitis B Virus Minichromosome C. Thomas Bock 1 *, Susanne Schwinn 2 , Stephen Locarnini 3 , Janet Fyfe 3 Michael P. Manns 1 , Christian Trautwein 1 and Hanswalter Zentgraf 2 1 Department of Gastroenterology and Hepatology, Medical School Hannover, Hannover, Germany 2 Applied Tumor Virology German Cancer Research Center, Heidelberg, Germany 3 Victorian Infectious Disease Reference Laboratory, North Melbourne, Australia The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approxi- mately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core pro- tein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our ®ndings, con®rmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein com- plexes by 10 %. From this model of the HBV minichromosome we pro- pose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner. # 2001 Academic Press Keywords: HBV; core protein; minichromosome; electron microscopy; nucleosomal spacing *Corresponding author Introduction In differentiated cells of higher eukaryotes, most of the DNA is organized by histone and non-his- tone proteins into nucleosomal and supra-nucleo- somal structures. The architecture of the nucleosome has been the subject of several recent reviews. 1±5 Important functions of the nucleosomal structure are (i) to compact DNA, (ii) to act as a regulator of transcription, and (iii) to play an active role in transcriptional events. 6±11 Organization of DNA into a nucleosomal struc- ture has also been observed for the polyoma viruses, 12 simian virus 40 (SV40), 13 human (HPV), 14 and bovine papilloma viruses (BPV), 15 and hepad- na viruses. 16,17 These viruses all produce small covalently closed, circular double-stranded DNA (cccDNA) which is found in the nucleus of infected cells as a viral minichromosome. By using electron microscopic spread preparations, the viral mini- chromosome can be observed to form typical ``beads-on-a-string'' structures, revealing the nucleosomal packaging of transcriptionally inactive chromatin. 18,19 The nucleosomal spacing (repeat length) is speci®c for individual organisms, cell types and their developmental stages and spans from 160 bp to 240 bp. 20 ± 26 Even within the same cell, transcrip- tional activity can alter the repeat length of speci®c genes. 27 ± 29 However, the mechanisms by which altered repeat lengths are generated is poorly understood. It has been suggested that nucleosome positioning plays a role in gene activation by chan- Present address: C. Thomas Bock, Department of Pathology, Molecular Pathology, University of Tuebingen, Tuebingen, Germany. Abbreviations used: HBV, hepatitis B virus; NPC, nucleoprotein complex; ds, double-stranded; pd, partially double-stranded, cccDNA, covalently closed, circular DNA; HBcAg, hepatitis B virus core antigen; HBxAg, hepatitis B virus X-antigen; PBS, phosphate- buffered saline. E-mail address of the corresponding author: Thomas.Bock@med.uni-tuebingen.de doi:10.1006/jmbi.2001.4481 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 307, 183±196 0022-2836/01/010183±14 $35.00/0 # 2001 Academic Press