In vitro model for the nuclear transport of the hepadnavirus genome

Abstract

Hepadnaviruses contain a DNA genome, but they replicate via an RNA intermediate, synthesized by the cellular RNA polymerase II in the nucleus of the infected cell. Thus, nuclear transport of the viral DNA is required in the viral life cycle. Protein-free DNA is only poorly imported into the nucleus, so one or more of the viral proteins must be involved in the transport of the viral genome. In order to identify these viral proteins, we purified woodchuck hepadnavirus (WHV) core particles from infected woodchuck liver, isolated WHV DNA, and extracted the covalent complex of viral polymerase from the particles using urea. Intact core particles, the polymerase-DNA complex, or protein-free WHV DNA from core particles was added to digitonin-permeabilized HuH-7 cells, in which the cytosol was substituted by rabbit reticulocyte lysate (RRL) and an ATP-generating system. The distribution of the viral genome was analyzed by semiquantitative PCR or by hybridization in total nuclei, RRL, nuclear membranes, and nucleoplasm. The polymerase-DNA complex was efficiently transported into the nucleus, as indicated by the resistance of the nucleus-associated DNA to a short-term treatment with DNase I of the intact nuclei. The DNA within core particles stayed mainly in the cytosol and remained protected against DNase I. A minor part of the encapsidated DNA was bound to nuclei. It was protected against DNase I but became accessible after disruption of the nuclei. Deproteinized viral DNA completely remained in the cytosol. These data show that the viral polymerase is probably sufficient for mediating the transport of a hepadnavirus genome into the nucleus and that the viral core particles may release the genome at the nuclear membrane.