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 3D pol ) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3D pol 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 3D po1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.Rhinovirus infections are the leading cause of the common cold (35). There are over 100 serotypes of human rhinovirus (HRV), the majority of which utilize intercellular adhesion molecule 1 as their host-cell receptor. HRV serotype 16 (HRV16) from this major receptor group is a suitable virus for clinical study (26) and can serve as a model for studying the transmission of colds and virus-induced asthma (6). Rhinoviruses are positivestrand, nonenveloped RNA viruses and are members of the Picornaviridae family, which comprise one of the largest families of viral pathogens responsible for many serious human and animal diseases including the common cold, hepatitis, poliomyelitis, and foot-and-mouth disease.HRV16 contains a genome comprised of 7,124 nucleotides and includes a 5Ј nontranslated region, a single open reading frame, a 3Ј nontranslated region, and a poly(A) tail of approximately 40 nucleotides. The open reading frame encodes a single polyprotein of 2,152 residues (

Interdomain Communication in Hepatitis C Virus Polymerase Abolished by Small Molecule Inhibitors Bound to a Novel Allosteric Site

“…Consistently, mutation of Leu-30 to Ser or Arg results in an inactive enzyme (33). Helical regions at the tip of fingertip loops connecting the fingers and thumb are present in other viral RdRps (12)(13)(14)(15)(16)(17)19). The similarities in the structure and enzymatic mechanism (34) between NS5B and these polymerases suggest that flexibility of the fingertip loops could also be a feature of some of these RdRps, and inhibitor binding sites similar to the one described here could also be available for them.…”

“…As a result, the polymerase has a relatively closed and spherical appearance, and the active site cavity, to which the RNA template and the NTP substrates have access via two positively charged tunnels, is completely encircled. Structural studies have shown that other viral RdRps, such as those of poliovirus (12,13), human rhinovirus (14,15), bovine viral diarrhea virus (16), rabbit hemorrhagic disease virus (17), reovirus (18), and bacteriophage 6 (19), have the same global architecture and a similar closed conformation. Crystal structures of NS5B bound to a short RNA template or to NTPs have also been solved (20,21).…”

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

“…K276 is located at the top of the middle finger and is near the RNA template entry channel, but it does not directly interact with the RNA in any of the picornaviral 3D pol -RNA complexes whose structures have been solved thus far. The structural orientations of the phylogenetically conserved amino acids implicated in the polyadenylation of viral RNA are similar in the atomic structures of 3D pol of enterovirus 71 (35), a group A enterovirus; 3D pol of coxsackievirus B3 (36,37), a group B enterovirus; 3D pol of poliovirus (13,16), a group C enterovirus; 3D pol of rhinovirus type 16 (38,39), species A; 3D pol of rhinovirus type 14 (39), species B; and foot-and-mouth disease virus 3D pol (40,41). These phylogenetically conserved sequences and structures likely contribute to the reiterative transcription of poly(A) and poly(U) sequences during viral RNA replication in these other picornaviruses as well, thereby regulating the lengths of poly(A) at the 3= end of viral RNA.…”

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

“…Concerning the VPg-binding site on 3D pol , two different sites seem to coexist. The first complete crystal structures of Picornaviridae RdRps of various human rhinovirus (HRV) strains (1,32) showed a widely open access to the active site from the "front" of the RdRp. Accordingly, a model was proposed that accommodated the VPg primer in a "frontloading" position (1).…”

“…The first complete crystal structures of Picornaviridae RdRps of various human rhinovirus (HRV) strains (1,32) showed a widely open access to the active site from the "front" of the RdRp. Accordingly, a model was proposed that accommodated the VPg primer in a "frontloading" position (1). This was indeed demonstrated by the crystal structure of the FMDV RdRp in complex with VPg-pU (12).…”