IN-VITRO MODEL FOR THE NUCLEAR TRANSPORT OF THE HEPADNAVIRUS GENOME

Hepadnaviruses contain a DNA genome, but they replicate via an RNA int ermediate, synthesized by the cellular RNA polymerase II in the nucleu s of the infected cell. Thus, nuclear transport of the viral DNA is re quired in the viral life cycle. Protein-free DNA is only poorly import ed into the nucleus, so one or more of the viral proteins must be invo lved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particle s from infected woodchuck liver, isolated WHV DNA, and extracted the c ovalent complex of viral polymerase from the particles using urea, Int act core particles, the polymerase-DNA complex, or protein-free WHV DN A from core particles was added to digitonin-permeabilized HuH-7 cells , in which the cytosol mas substituted by rabbit reticulocyte lysate ( RRL) and an ATP-generating system. The distribution of the viral genom e was analyzed by semiquantitative PCR or by hybridization in total nu clei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA comp lex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment wit h DNase I of the intact nuclei. The DNA within core particles stayed m ainly in the cytosol and remained protected against DNase I. A minor p art of the encapsidated DNA was bound to nuclei. It was protected agai nst DNase I but became accessible after disruption of the nuclei. Depr oteinized viral DNA completely remained in the cytosol. These data sho w that the viral polymerase is probably sufficient for mediating the t ransport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.