Poliovirus RNA synthesis utilizes an RNP complex formed around the 5′-end of viral RNA.

Abstract

The structure of a ribonucleoprotein complex formed at the 5′‐end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf‐like structure and interact with both uncleaved 3CD, the viral protease‐polymerase precursor, and a 36 kDa ribosome‐associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem‐loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site‐directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic‐protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5′‐end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

A “Slide-back” Mechanism for the Initiation of Protein-primed RNA Synthesis by the RNA Polymerase of Poliovirus

“…5 AAA 7 Sequence of cre(2C) Elements on Replication Phenotypes-Based on mutational analyses of the HRV14cre(VP1) (19) and poliovirus cre(2C) (20) and in comparison with other known (17) or putative entero-and rhinovirus cre elements, a common 1 RXXX-AARXXXXXXR 14 motif has been proposed that is essential for VPg uridylylation in vitro. For poliovirus this motif is 1 GXXX-AAAXXXXXXA 14 ( Fig. 1A) in which the 5 AAA 7 triplet is likely to serve as template for VPg-uridylylation (10,11).…”

“…2 and 7). The viral proteins most directly involved in RNA synthesis are the template-and primer-dependent RNA polymerase 3D pol (8,9), the terminal protein VPg (9,10), and 3CD pro (10,11), a proteinase (12,13), and an important RNA-binding protein (14). The RNA polymerase has two distinct types of activities.…”

“…Second, it can covalently link a nucleotide to the hydroxyl group of tyrosine in VPg (9). The cis-replicating elements include a cloverleaf like structure in the 5Ј-NTR (14) and a heteropolymeric region in the 3Ј-NTR with the poly(A) tail (15). In addition, an important RNA hairpin (cre(2C)) is located in the PV-coding sequence of protein 2C…”

Binding of a cellular factor to the 3' untranslated region of the RNA genomes of entero- and rhinoviruses plays a role in virus replication.

“…The CL has also been shown to bind to a cellular factor of 36 kDa (p36), which was observed to promote binding of the viral protein 3CD (Andino et al, 1993). Recently, UV cross-linking experiments have suggested that a cellular factor(s) of a similar size (approximately 34-36 kDa) binds to the 3h UTR and is found at increased levels in infected cells (Roehl & Semler, 1995).…”

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.…”