Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer

Abstract

Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-A resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

FIG. 1.

(A) The averaged, solvent-flattened 2.6-Å electron density map calculated by using SAD phases and then superimposed on the final model in the region of motif C. The conserved Gly-Asp-Asp sequence is labeled. (B) A stereoview of the Cα trace for HRV16 3D^pol. The color of the trace corresponds to the N-terminal region (yellow), the fingers subdomain (red), the fingers insertion (purple), the palm subdomain (green), and the thumb subdomain (blue). N and C termini as well as every 20th residue are labeled accordingly.

FIG. 2.

(A) A ribbon representation of HRV16 3D^pol. Flat coils (α-helices), arrows (β-strands), and thin round coils (turns and loops) indicate secondary structural elements. The ribbon is colored according to scheme described in the legend of Fig. 1B. Catalytic Asp residues 327 and 328 are shown as stick models, while N and C termini are labeled accordingly. (B) Viral RdRp homology. The structures of PV 3D^pol (residues 12 to 37, 67 to 97, 181 to 266, and 291 to 461), RHDV RdRp, HCV NS5B, BVDV NS5B, and the bacteriophage Φ6 RdRp are shown in ribbon representations and colored by region based the scheme described in the legend of Fig. 1B.

FIG. 3.

(A) A stereoview of the N-terminal region tethering the fingers and thumb subdomains. The N-terminal residues (1 to 53) are shown as a yellow ribbon with selected side chains shown as stick models. The putative RNA binding cleft formed by the palm (green), fingers (red), and thumb (blue) subdomains is indicated. Phe 30 and Arg 49 are labeled for the purpose of sequence orientation. (B) The conserved polymerase structural motifs mapped onto HRV16 3D^pol. Motif A (red; residues 227 to 242), motif B (green; residues 290 to 307), motif C (yellow; residues 318 to 335), motif D (light blue; residues 227 to 242), motif E (dark blue; residues 368 to 377), and motif F (magenta; residues 167 to 176) are indicated. The putative metal binding residues (Asp 327 and Asp 328 from motif C) and the basic residues believed to interact with the rNTP (Lys 167 and Arg 174 from motif F) are indicated by stick models.

FIG. 4.

(A) A stereoview of the thumb subdomain rotation observed between molecules A (blue) and C (red). The molecules were aligned exclusively by the finger and palm subdomains (gray in both molecules). (B) Active site comparison between HRV16 3D^pol (green sticks), PV 3D^pol (blue sticks), and HCV NS5B (orange sticks). UTP (yellow sticks), magnesium ions (cyan spheres), and the 3′ end of the primer strand (beige sticks) from the rough HRV16 3D^pol-substrate model are also included. Residues are numbered according to the HRV16 3D^pol sequence.

FIG. 5.

Hypothetical elongation complex. (A) A stereoview of HRV16 3D^pol structure rendered as a surface and viewed from the rear of the molecule towards the rNTP binding tunnel. The protein is colored by region according to the scheme described in the legend of Fig. 1B. The template strand (gray), primer strand (gold), UTP (stick model), and metal ions (cyan spheres) are modeled into the enzyme active site. The 5′ template (T) overhang enters from the top of the polymerase. The incoming UTP and magnesium ions are positioned above Asp 327 of motif C. (B) Stereoview looking down on top of HRV16 3D^pol. The template strand (T), primer strand (P), and protein are colored as above. (C) Two loops of the fingers subdomain (residues 102 to 108 and residues 131 to 138) may interact with the duplex product. (D) Residues (408 to 420) from the thumb subdomain and motif E may help grip the 3′ end of the primer strand.

FIG. 6.

Stereoviews of hypothetical VPg uridylylation. (A) Viewed from the rear of the polymerase towards the rNTP binding tunnel, the location of residues implicated in PV 3AB binding (magenta sticks) (31) are mapped onto the thumb subdomain (blue) of a ribbon representation of HRV16 3D^pol. A schematic representation of a CRE-like stem-loop (gray) and a small peptide representing VPg (gold) are modeled to illustrate the front-loading VPg uridylylation mechanism. VPg-Tyr3, UTP, and Asp 327 from HRV16 3D^pol are represented by stick models, while the magnesium ions are shown as cyan spheres. (B) Stereoview of a surface model of VPg uridylylation. The protein model is colored according to the scheme described in the legend of Fig. 1B and is viewed looking down on the top of the polymerase. The models for CRE and VPg are colored as in panel A.