Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

Abstract

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C pro and the viral polymerase 3D pol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3D pol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D pol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D pol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.Poliovirus (PV), a member of the Picornaviridae family of RNA viruses, must simultaneously perform many tasks inside a host cell for efficient and successful viral replication, and only a small number of gene products are responsible for these processes. The virus produces a single polyprotein that is cleaved by virally encoded proteases. Many of the viral proteins, including precursor proteins, play multiple roles in viral replication.The viral protein 3CD is a multifunctional precursor to the poliovirus protease 3Cpro and the RNA-dependent RNA polymerase 3Dpol . The 3CD molecule retains its ability to function as a protease but lacks polymerase activity (20). Its proteolytic functions include cleavages, resulting in the production of structural proteins VP0, VP1, and VP3 and nonstructural proteins 3AB, 3CD, 3C pro , and 3D pol . In many of these functions, 3CD serves as a better protease than 3C pro , suggesting that the 3D region of 3CD contributes to that activity (34).3CD is also a crucial component of the viral replication complex. The 5Ј-terminal region of the poliovirus genome adopts a cloverleaf structure to which 3CD can bind in the presence of the viral protein 3AB or the cellular protein PCBP2 (2, 35, 49). Both forms of this ribonucleoprotein complex appear to play important roles in inducing RNA synthesis (49). The initiation of RNA synthesis is primed by the addition of two uridines to the 22-amino-acid protein primer VPg (3B) at the side-chain hydroxyl of Tyr3. Positive-strand synthesis is thought to use an RNA hairpin structure as its template within the PV genome (37,42). One model suggests that this cisacting replication signal in the 2C-noncoding region of the PV genome, cre(2C), binds to 3CD and forms a mu...

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

“…This had been proposed to assure the correct positioning of motif-A residue Asp238 and in that way the correct conformation of the active site in the palm subdomain (66). Nevertheless, the recent structure of PV 3CD pro has shown that the conformation of the active site, including Asp238 is preformed even when the N terminus is not in its final position (36). A similar scenario may be assumed for CVB3 3CD pro and 3D pol .…”

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

“…The picornaviral polymerases are unique in that they only become active upon complete proteolytic processing of the precursor 3CD pro (Andino et al, 1993;Thompson and Peersen, 2004;Marcotte et al, 2007). The structure of the poliovirus RdRp revealed that the N-terminal glycine residue of the fully processed polymerase is buried in a pocket at the base of the "fingers" domain, where it involves in four hydrogen bonds that effectively reposition Asp238 into the active site.…”

Picornavirus Genome Replication

“…Picornaviral 3CD protein is a fusion between 3C protease and 3D polymerase domains. Protein 3CD exhibits protease activity with a specificity and cata-lytic efficiency that is different from 3C but lacks polymerase activity, although the overall fold of the 3D domain of 3CD is quite similar to that of 3Dpol (15,16). In addition to protease activity, the 3C domain alone and in the context of 3CD exhibits both specific and nonspecific RNA binding activity (12, 13, 17-34).…”