A novel cis-acting element facilitates minus-strand DNA synthesis during reverse transcription of the hepatitis B virus genome

Abstract

Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5' end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3' end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the beta5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the beta5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the beta5 region into its own position in a mutant in which the sequences including the beta5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the beta5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.

FIG. 1.

Model for minus-strand transfer during hepadnaviral reverse transcription. The pgRNA with cis-acting elements relevant to minus-strand transfer is shown. The pgRNA contains direct repeat sequences of 11 nt, designated DR1 and DR2. DR1 is present twice in the pgRNA due to terminal redundancy (R); the one near the 3′ end is designated DR1*. The encapsidation signal (ɛ) near the 5′ terminus, which folds into a stem-loop structure, is shown. The P protein (shaded oval), serving as a primer, synthesizes the first 4 nt of minus-strand DNA by using the UUCA motif within the bulge of the stem-loop structure as a template. The P protein covalently linked to the first 4 nt of minus-strand DNA is transferred to the UUCA motif at DR1*, which is referred to as the acceptor.

FIG. 2.

A mutant with a deletion of the sequence between DR2 and DR1* is defective in minus-strand DNA synthesis. (A) Map of deletion mutants. The structure of pgRNA is shown at the top. E, EcoRI restriction site (nt 3182 to 3180). The nucleotide sequences that were deleted are depicted by shaded lines along with the deleted nucleotide numbers. A map of the R063 construct, a helper plasmid lacking 5′ ɛ, the encapsidation signal, is shown. (B) Southern blot analysis of the replication-intermediate DNAs extracted from cytoplasmic core particles. Huh7 cells were transfected with each deletion mutant or the wild type (WT), along with a helper plasmid (R063) providing the C and P proteins, and the viral replication intermediates were extracted as described in Materials and Methods. Two restriction fragments, of 3.3 and 2.0 kb, were employed as size markers (SM). The positions of RC DNA, DL DNA, and ssDNA, in order from the top, are indicated by open arrowheads. The fast migrating DL DNA found in the deletion mutants is indicated by closed arrowheads. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) RPAs were performed to measure the encapsidation efficiency of the deletion mutants. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper and the pCMV-lacZ/30 plasmid as an internal control (10). The protected RNA fragments derived from HBV RNA and the lacZ transcript are indicated by closed and open left-facing arrowheads, respectively. The probes used for the analysis of HBV RNA and the lacZ RNA are indicated by closed and open right-facing arrowheads, respectively. Only one-third of the RNAs extracted from total cells (T) were loaded relative to those of capsids (C). (D) Viral DNA synthesis relative to the wild type after normalization to the amount of encapsidated RNA. Error bars represent the standard deviations from six independent transfections.

FIG. 2.

A mutant with a deletion of the sequence between DR2 and DR1* is defective in minus-strand DNA synthesis. (A) Map of deletion mutants. The structure of pgRNA is shown at the top. E, EcoRI restriction site (nt 3182 to 3180). The nucleotide sequences that were deleted are depicted by shaded lines along with the deleted nucleotide numbers. A map of the R063 construct, a helper plasmid lacking 5′ ɛ, the encapsidation signal, is shown. (B) Southern blot analysis of the replication-intermediate DNAs extracted from cytoplasmic core particles. Huh7 cells were transfected with each deletion mutant or the wild type (WT), along with a helper plasmid (R063) providing the C and P proteins, and the viral replication intermediates were extracted as described in Materials and Methods. Two restriction fragments, of 3.3 and 2.0 kb, were employed as size markers (SM). The positions of RC DNA, DL DNA, and ssDNA, in order from the top, are indicated by open arrowheads. The fast migrating DL DNA found in the deletion mutants is indicated by closed arrowheads. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) RPAs were performed to measure the encapsidation efficiency of the deletion mutants. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper and the pCMV-lacZ/30 plasmid as an internal control (10). The protected RNA fragments derived from HBV RNA and the lacZ transcript are indicated by closed and open left-facing arrowheads, respectively. The probes used for the analysis of HBV RNA and the lacZ RNA are indicated by closed and open right-facing arrowheads, respectively. Only one-third of the RNAs extracted from total cells (T) were loaded relative to those of capsids (C). (D) Viral DNA synthesis relative to the wild type after normalization to the amount of encapsidated RNA. Error bars represent the standard deviations from six independent transfections.

FIG. 3.

A mutant lacking the β region synthesized a significantly reduced amount of minus-strand DNA initiated at DR1*. (A) Strategies for primer extension analysis to determine the 5′ ends of minus-strand DNAs produced by mutants lacking the β region. M2 and M3 primers with nucleotide numbers are depicted on the minus-strand DNA. The oval circle represents the P protein covalently linked to the 5′ end of the minus-strand DNA. (B) Primer extension analysis with the M2 primer. A primer extension analysis was performed to determine the 5′ termini of minus-strand DNAs produced by the deletion mutants. A sequencing ladder generated with the M2 primer flanks the extension reactions. DR1* (wild type) is denoted by a closed arrowhead. The position of the predicted DR1* site for the R035 mutant is denoted by an open arrowhead. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (C) Primer extension analysis with the M3 primer. A sequencing ladder generated with the M3 primer flanks the extension reactions. The extension product derived from DNAs initiated at the UUC at nt 813 is denoted by an arrowhead. The extension product labeled DR1* (wild type) was not directly determined by the sequencing ladder but by comparison with the data shown in Fig. 2B. Equal amounts of wild-type and mutant samples were analyzed. (D) Map of pgRNA showing UUC motifs. Vertical bars below the line depict the positions of numerous UUC motifs in the pgRNA. Arrows denote the acceptor sites at DR1* and the UUC at nt 813, respectively. Three restriction sites in the DNA genome are shown: B, BstEII; E, EcoRI; S, SphI.

FIG. 4.

The β region failed to exert its function in reverse orientation. (A) Map of the mutants. Two unique restriction sites, for PmlI (P) and HindIII (H), were introduced to facilitate the inversion of the β region. The R806, R808, and R809 mutants, with either one or both novel restriction sites, were intermediate constructs used to construct the R810 mutant, in which the β region was inserted in the reverse orientation, as indicated by a left-facing arrow. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) RNase protection analysis was performed as described in the legend for Fig. 2C.

FIG. 4.

The β region failed to exert its function in reverse orientation. (A) Map of the mutants. Two unique restriction sites, for PmlI (P) and HindIII (H), were introduced to facilitate the inversion of the β region. The R806, R808, and R809 mutants, with either one or both novel restriction sites, were intermediate constructs used to construct the R810 mutant, in which the β region was inserted in the reverse orientation, as indicated by a left-facing arrow. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) RNase protection analysis was performed as described in the legend for Fig. 2C.

FIG. 4.

The β region failed to exert its function in reverse orientation. (A) Map of the mutants. Two unique restriction sites, for PmlI (P) and HindIII (H), were introduced to facilitate the inversion of the β region. The R806, R808, and R809 mutants, with either one or both novel restriction sites, were intermediate constructs used to construct the R810 mutant, in which the β region was inserted in the reverse orientation, as indicated by a left-facing arrow. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) RNase protection analysis was performed as described in the legend for Fig. 2C.

FIG. 5.

Determination of the boundary of the β region by deletion analysis. (A) Map of seven deletion mutants and pgRNA. The nucleotide sequences deleted from each mutant are depicted by shaded boxes. The mutant names, along with the β-region nucleotide numbers deleted from each construct, are presented to the left. The PRE is indicated by a hatched box, along with two minimal essential elements, HSLα and HSLβ1 (26). (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) The β5 region is the most critical sequence for its function. A primer extension analysis was performed with the M2 primer to determine the 5′ termini of minus-strand DNAs synthesized by each mutant. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type is indicated. Faster migrating extension products detected for each deletion mutant correspond to the minus-strand DNA initiated at DR1* of each mutant. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (D) None of the β-region deletion mutants were defective in RNA processing and encapsidation. An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation. (E) Quantitative analysis of the total DNA synthesized normalized to the amount of encapsidated RNA for each mutant. Error bars represent standard deviations from four independent experiments.

FIG. 5.

Determination of the boundary of the β region by deletion analysis. (A) Map of seven deletion mutants and pgRNA. The nucleotide sequences deleted from each mutant are depicted by shaded boxes. The mutant names, along with the β-region nucleotide numbers deleted from each construct, are presented to the left. The PRE is indicated by a hatched box, along with two minimal essential elements, HSLα and HSLβ1 (26). (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) The β5 region is the most critical sequence for its function. A primer extension analysis was performed with the M2 primer to determine the 5′ termini of minus-strand DNAs synthesized by each mutant. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type is indicated. Faster migrating extension products detected for each deletion mutant correspond to the minus-strand DNA initiated at DR1* of each mutant. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (D) None of the β-region deletion mutants were defective in RNA processing and encapsidation. An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation. (E) Quantitative analysis of the total DNA synthesized normalized to the amount of encapsidated RNA for each mutant. Error bars represent standard deviations from four independent experiments.

FIG. 5.

Determination of the boundary of the β region by deletion analysis. (A) Map of seven deletion mutants and pgRNA. The nucleotide sequences deleted from each mutant are depicted by shaded boxes. The mutant names, along with the β-region nucleotide numbers deleted from each construct, are presented to the left. The PRE is indicated by a hatched box, along with two minimal essential elements, HSLα and HSLβ1 (26). (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) The β5 region is the most critical sequence for its function. A primer extension analysis was performed with the M2 primer to determine the 5′ termini of minus-strand DNAs synthesized by each mutant. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type is indicated. Faster migrating extension products detected for each deletion mutant correspond to the minus-strand DNA initiated at DR1* of each mutant. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (D) None of the β-region deletion mutants were defective in RNA processing and encapsidation. An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation. (E) Quantitative analysis of the total DNA synthesized normalized to the amount of encapsidated RNA for each mutant. Error bars represent standard deviations from four independent experiments.

FIG. 5.

Determination of the boundary of the β region by deletion analysis. (A) Map of seven deletion mutants and pgRNA. The nucleotide sequences deleted from each mutant are depicted by shaded boxes. The mutant names, along with the β-region nucleotide numbers deleted from each construct, are presented to the left. The PRE is indicated by a hatched box, along with two minimal essential elements, HSLα and HSLβ1 (26). (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) The β5 region is the most critical sequence for its function. A primer extension analysis was performed with the M2 primer to determine the 5′ termini of minus-strand DNAs synthesized by each mutant. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type is indicated. Faster migrating extension products detected for each deletion mutant correspond to the minus-strand DNA initiated at DR1* of each mutant. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (D) None of the β-region deletion mutants were defective in RNA processing and encapsidation. An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation. (E) Quantitative analysis of the total DNA synthesized normalized to the amount of encapsidated RNA for each mutant. Error bars represent standard deviations from four independent experiments.

FIG. 5.

Determination of the boundary of the β region by deletion analysis. (A) Map of seven deletion mutants and pgRNA. The nucleotide sequences deleted from each mutant are depicted by shaded boxes. The mutant names, along with the β-region nucleotide numbers deleted from each construct, are presented to the left. The PRE is indicated by a hatched box, along with two minimal essential elements, HSLα and HSLβ1 (26). (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the mutants. (C) The β5 region is the most critical sequence for its function. A primer extension analysis was performed with the M2 primer to determine the 5′ termini of minus-strand DNAs synthesized by each mutant. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type is indicated. Faster migrating extension products detected for each deletion mutant correspond to the minus-strand DNA initiated at DR1* of each mutant. Huh7 cells were cotransfected with each deletion mutant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and mutant samples were analyzed. (D) None of the β-region deletion mutants were defective in RNA processing and encapsidation. An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation. (E) Quantitative analysis of the total DNA synthesized normalized to the amount of encapsidated RNA for each mutant. Error bars represent standard deviations from four independent experiments.

FIG. 6.

Ectopic insertion of the β5 region is sufficient to restore the initiation of minus-strand DNA synthesis at DR1*. (A) Map of two variants with the heterologous lacZ sequence in place of the β region. In R825, the heterologous lacZ sequence substituted for the β region. The nucleotide sequence encoding the β5 region was inserted back into its own position in R826. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the variants. (C) Primer extension analysis with the M2 primer to determine the 5′ termini of minus-strand DNAs produced by the variants. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type and R826 is indicated by an arrowhead. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and variant samples were analyzed. (D) An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation.

FIG. 6.

Ectopic insertion of the β5 region is sufficient to restore the initiation of minus-strand DNA synthesis at DR1*. (A) Map of two variants with the heterologous lacZ sequence in place of the β region. In R825, the heterologous lacZ sequence substituted for the β region. The nucleotide sequence encoding the β5 region was inserted back into its own position in R826. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the variants. (C) Primer extension analysis with the M2 primer to determine the 5′ termini of minus-strand DNAs produced by the variants. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type and R826 is indicated by an arrowhead. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and variant samples were analyzed. (D) An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation.

FIG. 6.

Ectopic insertion of the β5 region is sufficient to restore the initiation of minus-strand DNA synthesis at DR1*. (A) Map of two variants with the heterologous lacZ sequence in place of the β region. In R825, the heterologous lacZ sequence substituted for the β region. The nucleotide sequence encoding the β5 region was inserted back into its own position in R826. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the variants. (C) Primer extension analysis with the M2 primer to determine the 5′ termini of minus-strand DNAs produced by the variants. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type and R826 is indicated by an arrowhead. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and variant samples were analyzed. (D) An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation.

FIG. 6.

Ectopic insertion of the β5 region is sufficient to restore the initiation of minus-strand DNA synthesis at DR1*. (A) Map of two variants with the heterologous lacZ sequence in place of the β region. In R825, the heterologous lacZ sequence substituted for the β region. The nucleotide sequence encoding the β5 region was inserted back into its own position in R826. (B) Southern blot analysis performed as described in the legend for Fig. 2B. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Only one-half of the DNAs extracted from the wild type were loaded relative to those of the variants. (C) Primer extension analysis with the M2 primer to determine the 5′ termini of minus-strand DNAs produced by the variants. A sequencing ladder generated with the M2 primer flanks the extension reactions. The acceptor site at DR1* utilized by the wild type and R826 is indicated by an arrowhead. Huh7 cells were cotransfected with each variant or the wild type, as indicated above each lane, along with the R063 plasmid as a helper. Equal amounts of wild-type and variant samples were analyzed. (D) An RPA analysis was performed as described in the legend for Fig. 2C to examine the efficiency of RNA processing and encapsidation.