Figure 1.
Key roles of the P protein - ε RNA interaction in HBV replication.
The line with the hairpin structures represents the terminally redundant pgRNA which also serves as mRNA for core protein and P protein; ε and the direct repeats DR1, DR2 and DR1* are indicated. Binding of P to ε initiates their co-encapsidation, and also protein-primed reverse transcription. In this priming reaction, the 3′ nucleotide of the ε bulge and/or the first nucleotide of the upper stem (termed A1 in this study) template the covalent addition of the first DNA nucleotide to a Tyr residue in the TP domain (not explicitly shown) and its extension by two or three nucleotides along the bulge. Upon translocation to a matching acceptor site in DR1* the oligonucleotide is extended into full-length minus-strand DNA, with concomitant degradation of the pgRNA. Subsequent plus-strand synthesis (not shown) eventually yields the relaxed circular (RC) DNA found in virions. The differing shapes of ε and P symbolize conformational alterations that are as yet only well established for DHBV.
Figure 2.
Predicted spacial location of the targeted upper stem residues and mutational design.
(A) Model for the 3D structure of the ε stem-loop. 3D structure prediction as implemented in the MC-Fold and MC-Sym program package [46] was combined with NMR-based structural information on the upper stem and apical loop ([39], PDB entry 2IXY) to derive a tentative model for the entire ε element with its lower stem (green), bulge (blue), upper stem (yellow) and apical loop (red). The same model is shown from two different angles to provide a visual impression of the spacial distribution of the targeted residues A1, A2, A9, A10, U13 and U15 (all in cyan). Their major groove location and close juxtaposition to the bulge and loop, respectively, is supported by the NMR data; other features, including the relative orientation of upper stem vs. lower stem, are arbitrary. (B) Specific mutations investigated. Nucleotide exchanges and their positions in a 2D representation of ε together with the designations of the mutants are indicated. A linear representation is shown in Table 1.
Figure 3.
Impact of individual ε mutations on viral DNA accumulation.
HepG2 cells were transfected with the wild-type (wt) HBV expression vector pCH-9/3091, or derivatives containing the mutant 5′ ε sequences shown in Fig. 2B. The + or − signs indicate whether canonical base-pairs could form between residues at the A1-U29 and A2-U28 positions; potential G-U pairs are separately indicated. Viral DNAs from cytoplasmic nucleocapsids were monitored by Southern blotting (top panel) using a 32P-labeled HBV DNA probe; M, 3.2 kb restriction fragment corresponding to a unit length double-stranded linear (dsL) HBV genome. As controls, core protein and β-actin mRNA levels in the source lysates were monitored by Western blotting (middle panel) and RT-PCR (lower panel). Numbers below each lane show the accumulation of viral DNA replicative intermediates, measured by phosphorimaging, relative to those produced by the wild-type HBV construct which was set to 100. Mean values ± standard deviation were derived from two independent experiments.
Figure 4.
Direct confirmation of low DNA content in intact capsids derived from mutant ε constructs.
(A) DNA detection by molecular hybridization. Cytoplasmic capsids from cells transfected with the indicated constructs were separated by NAGE. After blotting, HBV DNA in the capsids was monitored by hybridization with a minus-strand specific probe, and capsids by immunodetection (panel labeled capsids). β-actin mRNA as determined by RT-PCR (panel labeled β-actin) served as loading control. (B) Endogenous polymerase assays (EPAs). One aliquot each of cytoplasmic capsids was subjected to EPA conditions in the presence of α-32P-dATP or α-32P-dCTP, then separated by NAGE. Labeled products associated with the capsids were visualized by autoradiography. Y63F refers to a replication-defective HBV construct in which the priming Tyr63 residue of P was replaced by Phe. A third aliquot from each sample was used for immunodetection of NAGE-separated capsids (panel labeled capsids). (C) Relative EPA activities. The bar graph shows the signal intensities generated by individual mutants for α-32P-dCTP and α-32P-dATP EPAs relative to that produced by wild-type HBV which was set at 100. Numbers are mean values from at least two independent experiments; error bars indicate standard deviation.
Figure 5.
Impact on pgRNA encapsidation of selected mutants displaying reduced DNA accumulation.
Total cytoyplasmic RNA and capsid-associated RNA from cells transfected with the indicated constructs were analyzed by RNase protection assays via hybridization to a 314 nucleotide antisense riboprobe (indicated by an asterisk) containing 41 nucleotides of non-HBV sequence; RNase digestion is expected to yield a protected fragment of about 270 nt (indicated by the arrow). Numbers below each lane show the encapsidation efficiency, measured as the ratio of encapsidated versus total pgRNA, relative to that produced by the wild-type HBV construct which was set to 100. Mean values ± standard deviation are from three independent experiments.
Figure 6.
In vitro priming activities of selected ε RNAs showing reduced DNA accumulation in cells.
(A) Increased priming signals by co-expression of wild-type ε RNA and increased α-32P-dATP concentration. FLAG-tagged HBV P protein from cells transfected with only the P protein vector (lane P), or cotransfected with a wild-type ε RNA expression vector (lanes P + ε) was immobilized on anti-FLAG antibody beads. One third each of the immunoprecipitate was incubated with 1 µl or 2 µl of α-32P-dATP (3,000 Ci/mmol) as reported [44]; subsequently, the beads were boiled in SDS-PAGE sample buffer, and the released material was analyzed by SDS-PAGE and autoradiography. The remaining one third of the immunopellet was analyzed for FLAG-tagged P protein by Western blotting (panel anti-FLAG). (B) In vitro priming activities of selected ε RNA variants. FLAG-tagged P protein complexes with the indicated ε RNAs were expressed, affinity purified and subjected to in vitro priming conditions as in (A), using 2 µl α-32P-dATP. The molecular mass marker positions indicated on the left (in kDa) are approximations inferred from the respective marker protein positions on the SDS-PAGE gels used for the anti-FLAG immunoblots which were run in parallel under identical conditions. Numbers below the autoradiogram indicate mean signal intensities ± standard deviation from two independent experiments relative to that produced by the wild-type ε RNA complex which was set to 100.
Table 1.
Sequences of investigated ε mutants and summary of phenotypes.
Figure 7.
Models for the position-related functional impacts of mutations in the upper stem.
(A) Base-pairing at the A1/A2 - U28/U29 positions may indirectly affect formation of a priming-active template structure. Replacement by G of A1 (A1G) or A1 plus A2 (A1,2G; not shown) nearly abrogated in vitro priming and DNA accumulation, whereas DNA accumulation was largely restored via concomitant replacement by C of U29 (rb1GC) or U28 plus U29 (rb1,2GC); n.d., not determined. Sequence-dependent formation of non-productive alternative structures could explain these divergent phenotypes. The model implies a (not experimentally proven) cross-bulge pair between A1g and the 5′ terminal bulge C residue (highlighted by an oval) which impairs template utilization (upward pointing arrow). This would be disfavored by stabilizing the original base-pairing pattern, via one (rb1GC; equally favored g1-C or g1-U29 pairs) or better two (rb1,2GC; mostly g1-C29 plus g2-C28) pairs at the base of the upper stem. U residues at the A1 and A2 position (A1U; A1,2U) cannot form cross-bulge base-pairs (not shown) and do not induce defects in DNA accumulation. See text for further details. (B) Major position-related functional impacts of upper stem mutations. This conceptual model summarizes the divergent phenotypes of mutants with a negative impact on viral DNA accumulation. Negatively acting mutations at A1/A2 had no defect in pgRNA encapsidation, produced a wild-type-like pattern of DNA, however at greatly reduced levels, and showed low or no in vitro priming. This suggests a major impact on priming efficiency per se (symbolized by the upward and downward arrows). Negatively acting mutations at A9/A10 did also not interfere with pgRNA packaging; however, only faster migrating DNA species were formed. Together with their clear in vitro priming activity, this implies a defect in primer translocation to the DR1* acceptor, e.g. via formation of an improper primer by improper initiation site selection (symbolized by the arrow pointing towards the template region). Negatively acting apical loop mutations reduced pgRNA encapsidation, displayed overall reduced DNA accumulation plus an excess of faster-migrating over full-length DNA, and showed low in vitro priming. This implies a general impact on P binding and/or formation of an encapsidation-proficient structure (symbolized by the curved arrows pointing towards P and core), combined with partial defects in protein priming efficiency and proper initiation site selection. See text for further details.