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Review
. 2015 Aug 3:206:12-26.
doi: 10.1016/j.virusres.2014.12.028. Epub 2015 Jan 12.

Initiation of protein-primed picornavirus RNA synthesis

Aniko V Paul  1 , Eckard Wimmer  2
Affiliations
Review

Initiation of protein-primed picornavirus RNA synthesis

Aniko V Paul et al. Virus Res. .

Abstract

Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. The Picornaviridae family constitutes a large group of plus strand RNA viruses that possess a small terminal protein (VPg) covalently linked to the 5'-end of their genomes. The RNA polymerases of these viruses use VPg as primer for both minus and plus strand RNA synthesis. In the first step of the initiation reaction the RNA polymerase links a UMP to the hydroxyl group of a tyrosine in VPg using as template a cis-replicating element (cre) positioned in different regions of the viral genome. In this review we will summarize what is known about the initiation reaction of protein-primed RNA synthesis by the RNA polymerases of the Picornaviridae. As an example we will use the RNA polymerase of poliovirus, the prototype of Picornaviridae. We will also discuss models of how these nucleotidylylated protein primers might be used, together with viral and cellular replication proteins and other cis-replicating RNA elements, during minus and plus strand RNA synthesis.

Keywords: Cis-replicating RNA element (cre); Picornavirus; RNA polymerase; RNA replication; Terminal protein VPg; Uridylylation of VPg.

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Figures

Fig. 1
Fig. 1
Initiation mechanisms of RNA synthesis by plus strand RNA viruses. Two basic types of mechanisms for RNA synthesis by plus strand RNA virus RNA polymerases are shown (162). (A) Primer-independent (de novo) initiation. De novo initiation involves starting an RNA chain, usually with a purine nucleoside triphosphate, templated by a pyrimidine at the 3’-end of the template strand or at an internal site. (B) Primer-dependent initiation. The primer is either an oligonucleotide or a protein. A 3’-hydroxyl group of a nucleotide or the hydroxyl group of a tyrosine or serine residue of a peptide/protein provides the hydroxyl group for the formation of a phosphodiester bond with the first nucleotide.
Fig. 2
Fig. 2
Genome structure of poliovirus and processing of the polyprotein. The RNA genome of poliovirus contains a long 5’ NTR, a single large open reading frame, and a short 3’NTR, which is terminated by a poly(A) tail. At its 5’-end the RNA is covalently linked to a small peptide called VPg. The polyprotein made during translation of the RNA contains one structural (P1) and two nonstructural precursors (P2, P3). The polyprotein is processed into precursor and mature proteins by proteinases 2Apro and 3Cpro/3CDpro. The maturation cleavage of VP0 into VP2 and VP4 occurs by an autocatalytic mechanism.
Fig. 3
Fig. 3
RNA structures involved in poliovirus RNA replication. There are three RNA structures in poliovirus RNA that are required or important for RNA replication (section 2.1) (131). A cloverleaf-like structure is located at the very 5’-end of poliovirus RNA. It contains the terminal UMP that is linked to the hydroxyl group of tyrosine in VPg. The amino acid sequence of VPg is shown below and the residues important for RNA replication are shown in red. The cloverleaf binds viral protein 3CDpro to stem-loop d and cellular protein PCBP2 to stem-loop b and a C-rich spacer. Viral protein 3AB also binds to stem loop b. The cre(2C) is a hairpin located in the coding sequence of 2CATPase. It is the template for the linkage of 2 UMPs to VPg during the initiation of RNA synthesis. The third RNA structure used during PV RNA replication is the 3’NTR, which contains two stem-loops, X and Y. These stem loops are involved in a “kissing” interaction (not shown). The binding of cellular poly(A) binding protein to the poly(A) tail is believed to be important for the circularization of the RNA genome.
Fig. 4
Fig. 4
Secondary structures of the picornavirus cre elements and their locations in the genome. (A) A simplified version of the enterovirus and rhinovirus RNA genome is shown on top. Open loops of the PV1, HRV2 and HRV14 cre elements (section 2.1.3) are shown in detail with G1 and A14 shown in blue and A5 and A6 that template VPgpUpU synthesis are shown in red. Only the upper part of the stem is shown. (B) A simplified version of the cardiovirus and aphthovirus RNA genomes is shown on top. The hepatovirus genome is the same except it lacks the L protein. The loops of the EMCV, FMDV, and HAV cre elements (section 2.1.3) are shown in detail and A5 and A6 that template VPgpUpU synthesis are shown in red.
Fig. 4
Fig. 4
Secondary structures of the picornavirus cre elements and their locations in the genome. (A) A simplified version of the enterovirus and rhinovirus RNA genome is shown on top. Open loops of the PV1, HRV2 and HRV14 cre elements (section 2.1.3) are shown in detail with G1 and A14 shown in blue and A5 and A6 that template VPgpUpU synthesis are shown in red. Only the upper part of the stem is shown. (B) A simplified version of the cardiovirus and aphthovirus RNA genomes is shown on top. The hepatovirus genome is the same except it lacks the L protein. The loops of the EMCV, FMDV, and HAV cre elements (section 2.1.3) are shown in detail and A5 and A6 that template VPgpUpU synthesis are shown in red.
Fig. 5
Fig. 5
“Slide-back” model for VPgpUpU synthesis during initiation of RNA synthesis. The upper stem of cre(2C) interacts with 2 molecules of 3CDpro (or 3Cpro) (section 3.7). 3CDpro binds VPg with the back side of its 3Dpol domain, where another molecule of 3Dpol links UMP to the hydroxyl group of tyrosine in VPg. A5 in the loop of cre(2C) is the template for the linkage of the first UMP (U1) to VPg yielding VPgpU1 . VPgpU1 “slides-back” to hydrogen bond with A6 and the second UMP (U2) is templated again by the A5 nucleotide during the elongation step yielding VPgpU1U2. The nucleotides involved in the “slide back” are boxed. Nucleotides A5 and A6 of the conserved motif are shown in bold.
Fig. 6
Fig. 6
Models of minus and plus strand RNA synthesis. (A) The poliovirus RNA circularizes through the interaction of the cloverleaf/PCBP2/3CDpro complex with PABP that is bound to the poly(A) at the 3’-end of the genome (section 4). VPgpUpU is synthesized on the cre(2C) and is probably transferred in a complex by 3Dpol and/or 3CDpro to the 3’-end of the poly(A) tail. There it serves as the primer for minus strand RNA synthesis yielding a double-stranded replicative form (RF). (B) The 5’-end of the RF is destabilized by the binding of viral and cellular proteins. 3CDpro in a complex with PCBP2 or 3AB binds to the plus strand cloverleaf after destabilizing the 5’-end of the RF. 2CATPase and hRNP C bind to and stabilize the minus strand cloverleaf (section 4). VPgpUpU that was made prior to or during minus strand synthesis is transferred in a complex with 3Dpol and or 3CDpro to the 3’-terminal two As of the minus strand. There VPgpUpU is elongated by 3Dpol yielding a replicative intermediate (RI) that contains multiple plus strands at various stages of elongation. The final products are the full length VPg-linked plus strands.
Fig. 6
Fig. 6
Models of minus and plus strand RNA synthesis. (A) The poliovirus RNA circularizes through the interaction of the cloverleaf/PCBP2/3CDpro complex with PABP that is bound to the poly(A) at the 3’-end of the genome (section 4). VPgpUpU is synthesized on the cre(2C) and is probably transferred in a complex by 3Dpol and/or 3CDpro to the 3’-end of the poly(A) tail. There it serves as the primer for minus strand RNA synthesis yielding a double-stranded replicative form (RF). (B) The 5’-end of the RF is destabilized by the binding of viral and cellular proteins. 3CDpro in a complex with PCBP2 or 3AB binds to the plus strand cloverleaf after destabilizing the 5’-end of the RF. 2CATPase and hRNP C bind to and stabilize the minus strand cloverleaf (section 4). VPgpUpU that was made prior to or during minus strand synthesis is transferred in a complex with 3Dpol and or 3CDpro to the 3’-terminal two As of the minus strand. There VPgpUpU is elongated by 3Dpol yielding a replicative intermediate (RI) that contains multiple plus strands at various stages of elongation. The final products are the full length VPg-linked plus strands.
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