Reconstitution of bluetongue virus polymerase activity from isolated domains based on a three-dimensional structural model

Abstract

Bluetongue virus (BTV) is a double-stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Currently there is no structural information available for VP1. By manual alignment of BTV, Reovirus and other viral RdRps we have generated a model for the structure of VP1, the RdRp of BTV. The structure can be divided into three domains: an N-terminal domain, a C-terminal domain, and a central polymerase domain. Mutation of the putative catalytic site in the central polymerase domain by site-directed mutagenesis abrogated in vitro replicase activity. Each of the domains was expressed individually and subsequently partially purified to obtain direct evidence for the location of polymerase activity and the nucleoside triphosphate binding site. The nucleoside triphosphate binding site was located by showing that CTP only bound to the full-length protein or to the polymerase domain and not to either of the other two domains. None of the domains had catalytic activity when tested individually or in tandem but when all three domains were mixed together the RdRp activity was reconstituted. This is the first report of the reconstitution of a functional viral RdRp in vitro from individual domains.

Figure 1

Alignments used to generate the polymerase domain model of BTV VP1. The alignment of BTV VP1 polymerase domain with poliovirus RdRp (A) or with RHDV RdRp (B). Pairwise alignments derived from the original alignment of all three sequences are shown for clarity. Identical residues are on a red background, and similar residues are in red font. Similarity was defined using ESPript 2.2, with a similarity score of 0.8 and using physicochemical properties of side chains as the criteria for similarity. Motif A is indicated with asterisks. Motif C is underlined.

Figure 2

Model of the polymerase domain of VP1 and comparison of the modeled structure at the conserved motifs A–D with the solved structures of other polymerases. (A) The polymerase domain model is based on the complete X‐ray crystal structures of the RdRps of two ssRNA viruses, poliovirus and RHDV. The “fingers” subdomain is in blue, the “palm” subdomain is in red and the “thumb” subdomain is green. Motif C in the “palm” subdomain is in yellow. (B) Expansion of the boxed region in panel A, showing the motif C (GDD motif) in yellow at residues 763–765. Amino acid side chains are depicted for motif C only. (C) A comparison of the model of BTV VP1 proximal to motifs A–D with the corresponding regions of the solved structures of RdRps. The regions of structure that correspond to the sequence motifs A–D are circled. Motif C is colored yellow. (i) modeled BTV VP1; (ii) RHDV RdRp; (iii) Reovirus λ3, and (iv) φ6 P2.

Figure 3

Modeled and cloned regions of BTV VP1. (A) The NTD modeled from amino acid residues 1–373 is shown in cyan. The PD model covers amino acid residues 581–880. The “fingers” subdomain is blue, the “palm” subdomain is red, and the “thumb” subdomain is green. The CTD modeled from amino acid residues 847–1295 is in magenta. The CTD model overlaps with the “thumb” subdomain of the PD model.

Figure 4

Alignment of structurally equivalent residues between the NTD and CTD models and the corresponding regions of λ3. (A) Alignment of the NTD model of BTV VP1 with the structural template (reovirus λ3). (B) Alignment of the CTD model of BTV VP1 with the structural template. Similar and identical residues are shaded as described for Figure 1.

Figure 5

Alignment of structurally equivalent residues of the polymerase domains of BTV VP1 and reovirus λ3. Similar and identical residues are shaded as described for Figure 1. Secondary structure elements are colored as for Figure 2.

Figure 6

Purification of wild‐type and mutant VP1 protein and replicase activity. (A) SDS‐PAGE of purified wild‐type His‐VP1 and His‐VP1 DD_764–765AA mutant. Lanes 1 and 3, protein size markers (kDa); lane 2, VP1 mutant; lane 4, wild‐type VP1. (B) Replicase assay of wild‐type and mutant DD_764–765AA VP1 proteins. Lane 1 ³²P‐labeled BTV‐10 genomic dsRNA; lane 2, replicase assay products using His‐VP1 DD_764–765AA; lane 3, replicase assay products using wild‐type His‐VP1.

Figure 7

Expression and purification of VP1 fragments. (A) VP1 fragments were expressed in E.coli strain HMS174 (DE3) and the total cell extracts were electrophoresed on a 12% SDS‐polyacrylamide gel followed by staining with Coomassie blue. Lane 1, empty vector control; lane 2, NTD; lane 3, PD; lane 4, CTD. Lanes 5–7 are the Western blot of the equivalent gel developed using a commercial anti‐polyhistidine antibody; lane 5, NTD; lane 6, PD; lane 7, CTD. Arrowheads indicate positions of VP1 fragments. (B) Western blot of purified VP1 fragments electrophoresed on a 12% SDS‐polyacrylamide gel. E.coli. cells were lysed by sonication and the VP1 fragments subsequently partially purified by a standard cobalt affinity chromatography as described in the text. The lane marked “P” is the pellet from the lysed cells; S is the soluble lysate; and the lane marked “E” is the material eluted from the cobalt beads. Position of size markers in kDa indicated on the left side.

Figure 8

NTP labeling of VP1 fragments. Fragments were incubated with [α‐³²P] CTP and bound nucleotides were covalently coupled before the fragments were electrophoresed on a 12% SDS‐polyacrylamide gel. Lane 1, full‐length VP1, purified from insect Sf 9 cells; lane 2, Sf 9 cell extract; lane 3, NTD; lane 4, PD; lane 5, CTD; lane 6, bacterial cell extract. Arrows indicate the major ³²P‐CTP labeled bands. Position of size markers in kDa indicated on the left side.

Figure 9

Replicase activity of VP1 fragments. Each of the fragments was tested for replicase activity as described in the text. ³²P‐labeled BTV‐10 genomic dsRNA markers (lane1). Positive controls for the replicase assay were purified VP1 (lane 2) or partially purified VP1 (lane 3) which was at a similar concentration and purity to the fragments. The inclusion or omission of each of the VP1 domains is indicated by a “+” or “−”, respectively. One microgram of protein was used when assaying VP1 fragments individually (lanes 4–6), and 500 ng of each purified VP1 fragment was used when assaying mixtures of two or three domains (lanes 8–11). Lane 7 is an empty vector control and contains the contaminating E.coli proteins that were bound to and subsequently eluted from the cobalt resin during purification.