An interdomain RNA binding site on the hepadnaviral polymerase that is essential for reverse transcription

Abstract

The T3 motif on the duck hepatitis B virus reverse transcriptase (P) is proposed to be a binding site essential for viral replication, but its ligand and roles in DNA synthesis are unknown. Here, we found that T3 is needed for P to bind the viral RNA, the first step in DNA synthesis. A second motif, RT-1, was predicted to assist T3. T3 and RT-1 appear to form a composite RNA binding site because mutating T3 and RT-1 had similar effects on RNA binding, exposure of antibody epitopes on P, and DNA synthesis. The T3 and RT-1 motifs bound RNA non-specifically, yet they were essential for specific interactions between P and the viral RNA. This implies that specificity for the viral RNA is provided by a post-binding step. The T3:RT-1 motifs are conserved with the human hepatitis B virus and may be an attractive target for novel antiviral drug development.

Figure 1. Structural organization of P

A. The four domains of DHBV P and their boundaries are shown along with the location of T3, RT-1 and the mAb 6 and 11 epitopes. Black bars represent P sequences employed as synthetic peptides. B. miniRT2, an active truncation of P, is shown with the boundaries of the truncations.

Figure 2. RNA binding by wild-type and T3-mutant P

A. Co-precipitation RNA binding assay. Wild-type P or P(I179D/L180D) were translated in vitro in the presence of radiolabeled ε or ε-dlBulge RNA, P was immunoprecipitated, and co-precipitating RNAs were resolved by electrophoresis. B. RNA binding activity by derivatives of P with mutations in T3. Wild-type and T3-mutant P molecules were assayed for the ability to bind ε using the assay in panel A, and RNA binding was normalized to amount of P that was immunoprecipitated and the specific activities of the RNAs. Error bars are the standard deviation from three experiments. ε RNA was employed except where noted. C. Mutating the T3 motif ablates encapsidation. LMH cells were transfected with CDNA3.1 as a negative control or with wild-type or P(K182E/K183E) overlength DHBV expression vectors. Four days post-transfection intracellular DHBV core particles were harvested, the endogenous nucleic acids were removed, and P was detected by western blot. D. The T3 peptide does not directly inhibit the DNA polymerase active site. ³⁵S-labeled P was translated in the presence of ε (“Before”) or absence of ε (“After”), the peptides were added at the indicated concentrations, an aliquot of the mixture was removed to monitor translation efficiency, ε was added to the “after” reactions, and then a priming assay was performed employing [α³²P]dGTP. The samples were resolved by SDS-PAGE, translation efficiency was monitored by detecting ³⁵S-labeled P (top panel), and priming was detected as ³²P-labeling of P (bottom panel). The ³⁵S signal was blocked in the priming reactions by overlaying the gel with exposed X-ray film. Cx, cycloheximide included during translation. MBP, a peptide from myelin basic protein at 1.0 mM as a negative control.

Figure 3. The T3 peptide can inhibit priming by miniRT2 in the absence of chaperones

MiniRT2 was purified under denaturing conditions to remove the bacterial chaperones, refolded, and incubated with ε and [α³²P]dGTP in the presence or absence of the indicated synthetic peptides. The left panel shows coomassie blue stained miniRT2 following purification. The right panel shows the priming signal following incubation of miniRT2 with increasing concentrations of T3 or the irrelevant MBP peptide. Peptide concentrations are in mM.

Figure 4. T3 and RT-1 are conserved among the hepadnaviruses

Multiple sequence alignments of regions flanking T3 (A) and RT-1 (B). DHBV3 to RGHBV are avian hepadnaviruses and WHV to HBV are mammalian hepadnaviruses.

Figure 5. RT-1 is predicted to be on the surface of the reverse transcriptase domain

HBV RT-1 sequences were mapped onto the predicted structure of the HBV reverse transcriptase domain (Das, Xiong et al., 2001). Yellow, RT-1; green, thumb; red, palm; blue, fingers; white, nucleic acids.

Figure 6. Effects of mutations to T3 and RT-1 on RNA binding, priming, and exposure of the mAb6 epitope

A. The relative effects on binding to ε, priming DNA synthesis, and the exposure of the occluded mAb 6 epitope are shown for mutations to T3 and RT-1. All activities are normalized to the activity of wild-type P. Mutations with the predicted pattern of effects on the three activities are indicated with black dots. The error bars represent ± 1 standard deviation from 3-4 experiments. B. Representative priming data. C. Representative mAb6 exposure data. D. Representative RNA binding data. Note that panels B-C are composites from multiple independent experiments.

Figure 7. T3 and RT-1 peptides bind RNA

A. T3 and RT-1 peptides bind ε. Top: The peptides were bound to a nitrocellulose filter in a slot-blot apparatus and then ³²P-labeled ε RNA was passed through the filter and the filter was washed. T3-scramble and T3B-scramble are negative control peptides in which the sequences have been scrambled; UL13 and Pep1 are irrelevant peptides. Bottom: T3B and RT1C peptides were loaded onto a filter in amounts ranging from 10 pMol to 0.3 pMol and RNA binding to ³²P-labeled ε RNA was measured as in the top panel. B. T3 and RT-1 peptides bind nucleic acids non-specifically. Top: 1Filter binding assays were performed with T3 and RT-1 peptides and either ε or the biologically inactive ε-dlBulge RNA in the presence or absence of a 50-fold excess of non-radioactive yeast tRNA. Bovine serum albumen (BSA) was used as an irrelevant protein. Bottom: Filter binding assays were performed with T3 and RT-1 peptides and ³²P-labeled DHBV core gene RNA or double-stranded DNA as irrelevant nucleic acid probes.

Figure 8. Priming and RNA binding activities of miniRT2 and miniRT2 derivatives with lesions in T3 and RT-1

A. MiniRT2, miniRT2-T3m, and miniRT2-RT1m purified under native conditions. Note that the bacterial chaperones co-purify with miniRT2 under native conditions but they are removed when the purification is performed under denaturing conditions (compare Figs. 8A and 3). B. 1Mutating the T3 and RT-1 motifs of miniRT2 ablates DNA priming. Equal amounts of DHBV miniRT2 and its T3m and RT1m derivatives were assayed for their ability to prime DNA synthesis using either ε or its inactive derivative ε-dlBulge as templates. C. 1 RNA binding by miniRT2 is non-specific. RNA binding by equal amounts of miniRT2 and its T3m and RT1m derivatives was tested in a filter-binding assay employing ³²P-labeled ε and ε-dlBulge RNAs as probes in the presence or absence of a 50-fold excess of unlabeled tRNA competitor.

Figure 9. Model for the contribution of T3 and RT-1 to the initial events of reverse transcription

See text for details. The putative locations of the T3 and RT-1 motifs are shown in relationship to the position of the DNA polymerase active site (AS). The domains of P are shaded as in Fig. 1.