Figure 1.
DHBV replication in avian cells and HBV replication in human cells.
(A) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. (B) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.
Figure 2.
DHBV replication in human cells and HBV replication in avian cells.
(A) DHBV in HepG2 cells. (B) HBV in LMH cells. Labeling and abbreviations are as in the legend to Figure 1. The position of a major DNA product of about 2 kb in length derived from enhanced HBV splicing in LMH cells is indicated (spl).
Figure 3.
Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA.
Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. (A) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. (B) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently (Figure S5B), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).
Figure 4.
Intracellular distribution and nuclease sensitivity of viral DNAs.
HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). (A) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. (B) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.
Figure 5.
Core protein association of nuclear DHBV and HBV DNA.
Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. (A) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. (B) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7.
Figure 6.
Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility.
(A) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. (B) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. (C) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.
Figure 7.
Polymerase linkage status of cytoplasmic versus nuclear viral DNAs.
HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). (A) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. (B) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.
Figure 8.
Evidence for a fraction of completely uncoated, largely protein-free nuclear HBV RC-DNA.
HepG2 cells were transfected with the vector for surface-deficient HBV. IPs were performed as in Figure 5, except that DNA from both the immunopellets (pel) and the supernatants (sup) was analyzed; in addition, the samples were treated, or not treated, with PK prior to DNA extraction as indicated. Note the high proportion of rcDNA in the αHBc supernatant from the nuclear vs. cytoplasmic sample, and the likewise high proportion of rcDNA signals in the nuclear vs. cytoplasmic samples not treated with PK.
Figure 9.
Efficient plus-strand DNA extension does not induce uncoating and polymerase removal from rcDNA.
Nucleocapsids were obtained by detergent-stripping of virions from highly viremic sera and subjected to a 16 h endogenous polymerase reaction (EPR; +dNTP) as described [26]. (A) DHBV. After the EPR, viral DNA was prepared via phenol extraction with or without prior PK treatment. All of the non-treated and the indicated fractions of the PK-treated sample were loaded on a gel and analyzed by Southern blotting. A very long exposure plus contrast enhancement (right panel) revealed a faint band at the dlDNA but not the rcDNA position. (B) Comparison of electrophoretic mobility of transfection-derived and virion-derived HBV DNA. Cytoplasmic DNA from transfected HepG2 cells and virion DNA (prior to EPR) was treated as indicated, isolated via phenol extraction, and analyzed by Southern blotting. Note the faster mobility of most virion-derived DNAs, indicative of incompletely extended plus-strands, and the virtual absence of signal without prior PK treatment. (C) HBV EPR products. Nucleocapsids subjected to long-term EPR conditions were treated as indicated. The shift towards the positions of full-length rcDNA and dlDNA after the EPR indicated that the nucleocapsid-borne polymerase was active. Nearly all DNA was resistant towards MN. Even an overexposed autoradiogram (right panel) did not reveal a signal without prior PK treatment.
Figure 10.
Virus-specific differences between DHBV and HBV cccDNA formation.
The cartoon summarizes the steady-state levels of different rcDNA forms in human HepG2 cells transfected with surface-deficient viruses. They suggest specific steps that differentially affect the rate of cccDNA formation. Cytoplasmic rcDNA of both viruses was largely present in intact nucleocapsids, with <10% in protein-free form. The about four-fold higher nuclear import efficiency of DHBV vs. HBV nucleocapsids is not considered. The red x indicates absence of intact polymerase; the minus-strand 5′ end may consist of a hydroxyl (OH) or phosphate (encircled P) group, or still contain one or more amino acid residues (aa) from the polymerase. (A) DHBV. Nuclei contained ∼50% rcDNA plus ∼50% cccDNA. Most of the rcDNA was present in MN resistant, i.e. bona fide intact, capsids. The fraction of protein-free rcDNA in the nucleus (∼20%) was only modestly higher than in the cytoplasm. If stably encapsidated nuclear rcDNA is a precursor to cccDNA (a), uncoating is the only slow step. If stable intranuclear capsids are a dead-end product nuclear import is rate-limiting. (B) HBV. Nuclei contained ≤10% MN resistant full-length rcDNA and <5% cccDNA; ≥80% of the rcDNA were MN sensitive. Hence conversion into these forms is efficient. About one third of the sensitive rcDNA could be precipitated with anti-HBc antibody; based on its genome region-selective sensitivity against MN we propose this fraction to represent partially opened capsids. About two thirds of the rcDNA was not precipitated, consistent with complete release. Hence initiation of uncoating is efficient but complete uncoating appears to be slow. Roughly two thirds in either fraction were protein-free, indicating that ∼40% of the nuclear HBV rcDNA were completely uncoated and no more linked to intact polymerase. This implies a major block for HBV in a subsequent step towards cccDNA which does not operate for DHBV. Little if any evidence was found in support of pathway b which implies partial uncoating and polymerase removal to occur in the cytoplasm.