Fig 1.
The structure of the hPBV RdRP.
(a) Ribbon diagram of the hPBV RdRP crystal structure. The N- and C-terminal domains are colored in yellow and magenta, respectively. The core polymerase domain is shown in three different colors with the fingers in blue, the palm in red, and the thumb subdomain in green. The three key aspartic acid residues are displayed in teal and the flexible insertion loop structure is highlighted in orange. Additionally, a close-up is shown of the hPBV RdRP superimposed with the surface view of the oligonucleotide from the ϕ6 RdRP replication initiation complex (PDB ID 1HI0). The oligonucleotide is colored in pink and the Mn2+ ion is colored grey. (b) The seven conserved core polymerase domain motifs. The N- and C-terminal domains are omitted for clarity. Different motifs are colored according to the color keys shown in the figure. (c) Secondary structure assignment of the hPBV RdRP. Disordered regions are shown by dashed lines. α-helices and β-strands are represented by cylinders and arrows, respectively. The seven polymerase motifs are boxed and labeled sequentially as G, F, A, B, C, D, and E. The color scheme is the same as in (a).
Table 1.
PBV RdRP data collection and refinement statistics.
Fig 2.
Surface representation of the hPBV RdRP.
(a) The WT RdRP molecule is shown from the front end in a similar orientation as in Fig 1A and colored according to its electrostatic potential with positively charged regions in blue and negatively charged regions in red. (b-d) WT RdRP with the insertion loop removed to reveal channels connected to the active site. Three consecutive views are provided, front (b), top (c) and back (d), that show the product channel, the template entry channel, and the nucleotide diffusion channel, respectively. In the top view (c), a positively charged groove next to the template entry channel is highlighted by a white dotted oval. The red dotted circle illustrates the approximate position of the polymerase active site in (b-d).
Fig 3.
The structure and enzymatic activies of the hPBV WT and ΔLOOP RdRP.
(a) Ribbon diagram of the hPBV ΔLOOP RdRP crystal structure. The color scheme and the viewing orientation is the same as that used for the WT RdRP in Fig 1A. Below is a structural alignment between the WT (cyan) and the ΔLOOP (magenta) RdRPs. (b) The replication and transcription activity and the template specificity of the hPBV WT (left) and ΔLOOP (right) RdRPs. Three ssRNA and two dsRNA templates were used: (+) and (-)strands of the PBV genome segment 2 (PBV2+ and PBV-, respectively), (+)strand of the ϕ6 genomic segment S (s+), PBV dsRNA genome segment 2 (PBV2) and ϕ6 genomic dsRNA (ϕ6). (c) A schematic representation of the TNTase activity using either ssRNA (left) or dsRNA (right) as a substrate. (d) TNTase activity assays for the WT and the ΔLOOP RdRPs using either ϕ6 genomic dsRNA (left) or ϕ6 and PBV specific ssRNA substrates (right). The RdRPs used are indicated on the top and the RNA templates on the bottom (b and d). The mobility of the ϕ6 and hPBV-specific dsRNAs and ssRNAs are marked on the left (b and d).
Fig 4.
The hPBV RdRP insertion loop enables primer-independent RNA synthesis.
(a) A schematic representation of the de novo (left) and back-priming (right) initiation modes of the PBV RdRP displaying the effect of the heat-denaturation on the replication reaction products. (b) Native agarose gel electrophoresis of the replication reaction products of ϕ6 Δs+ ssRNA before and after denaturation as indicated below. (c) Labeling of the replication reaction products of ϕ6 Δs+ ssRNA with [γ-32P]-GTP in the initiation of RNA replication. The RdRPs applied are indicated at the bottom and the mobilities of the dsRNAs and ssRNAs are marked on the left (b and c).
Fig 5.
Transcription activity of the hPBV RdRP.
(a) Schematic drawings showing radiolabeled products expected from conservative (left) and semi-conservative transcription (right). (b) Transcription activity of the WT and the ΔLOOP RdRPs using virion derived ϕ6 genomic dsRNA. The expected position of the L, M, S dsRNA segments are indicated on the left. Both [α-33P] UTP (left) and [ү-32P] GTP (right) were used to label reaction products. The RdRPs applied are indicated at the top. (c) Transcription activity of the WT RdRPs using PBV2 dsRNA templates synthesized in vitro. Three dsRNA templates were included: the full-length PBV2 (i.e. PBV2), PBV2 without the first 33 base pairs (i.e. Δ1–33), and PBV2 without the first 645 base pairs (i.e. Δ1–645). For the quantitation (right), the gel band intensities were normalized by the length (bp) of the dsRNA molecules. The RNAs applied and their (-)strand 3’-end sequences are shown at the bottom. The mobilities of the dsRNAs are marked on the left (b and c).
Fig 6.
Co-expression of the hPBV RdRP and CP.
(a) TEM images of the purified hPBV VLPs for the WT CP (top) and the Δ45CP (bottom). The scale bar represents 50 nm. (b) SDS-PAGE analysis of the recombinant capsids of both the full-length and Δ45 CPs. (c) Co-expression of the hPBV CP and RdRP. Samples included are a prestained protein marker (lane 1), the soluble fraction of the cell lysate (lane 2), the Ni-NTA bound fraction (lane 3), and the purified VLPs (lane 4). Proteins were separated by SDS-PAGE and detected by Western blot using either anti-6xHis or anti-CP antibodies (two upper panels) or staining with Coomassie blue (lower panel). The molecular weights of the pre-stained marker proteins are indicated in kDa on the left.
Fig 7.
RNA binding by the hPBV WT (a, c) and ΔLOOP (b, d) RdRPs analyzed by gel shift assays. The fraction of RNA bound was quantitated and plotted as a function of protein concentration. The values were normalized by the total amount of RNA. (a, c) Three distinct RNA oligonucleotides were used, including: (1) the first 20 nucleotides of the 5’-(+)UTR of hPBV genome segment 2 (black); (2) the last 20 nucleotides of the 3’-(+)UTR of the hPBV genome segment 2 (red); and (3) a 20-mer nonsensical CA repeat (blue). (b, d) Two RNA molecules derived from the PBV2+ were used, including the full-length PBV2+ and the PBV2+(Δ1–645). The obtained dissociation constants (Kd) for the different RNA oligonucleotides are indicated on the right.
Fig 8.
Proposed model for hPBV RdRP replication (left) and transcription (right) inside the viral capsid.
The hPBV RdRP molecules are displayed in green. The blue and red lines correspond to the (+) and (-)strand respectively with the nascent (+)strand RNA represented in purple (right). The 5’-terminal stem loop structure is displayed in yellow. How exactly the two ssRNA molecules interact with each other and also with the viral CP during assembly and replication is not yet clear, as indicated by the questions marks in the figure on the left. The parental (+)strand RNA is separated from the template RNA during transcription and directed towards a pore in the viral capsid (right).