RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms

Ferrer-Orta, Cristina; Ferrero, D.S.; Verdaguer, N.

doi:10.3390/v7082829

Cited by 58 publications

(97 citation statements)

References 91 publications

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“…This palm domain-based closure mechanism differs from what is observed in other classes of replicative polymerases where the palm is fully structured prior to NTP binding, and the nascent base pair is delivered into the active site for catalysis via molecular motions originating in the finger domain (2,3). Despite these different molecular motions, the structural end points are essentially equivalent, and the RdRPs retain the highly conserved polymerase active site geometry with aspartate residues and a magnesium-catalyzed twometal reaction mechanism (4,5). The origin of the palm-based movement in the viral RdRPs is likely the conserved molecular contact between the finger and thumb domains that stabilizes the protein structure at the expense of reducing finger domain flexibility (6).…”

mentioning

confidence: 89%

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

McDonald

Block

Beaucourt

et al. 2016

Journal of Biological Chemistry

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Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stoppedflow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg 2؉ ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe 364 and Pro 357 , which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe 364 to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.The RNA-dependent RNA polymerases (RdRPs) 2 from positive strand RNA viruses close their active sites for catalysis via a subtle NTP-induced conformational change within conserved motifs A and C (1). This palm domain-based closure mechanism differs from what is observed in other classes of replicative polymerases where the palm is fully structured prior to NTP binding, and the nascent base pair is delivered into the active site for catalysis via molecular motions originating in the finger domain (2, 3). Despite these different molecular motions, the structural end points are essentially equivalent, and the RdRPs retain the highly conserved polymerase active site geometry with aspartate residues and a magnesium-catalyzed twometal reaction mechanism (4, 5). The origin of the palm-based movement in the viral RdRPs is likely the conserved molecular contact between the finger and thumb domains that stabilizes the protein structure at the expense of reducing finger domain flexibility (6).One key characteristic of the viral RdRPs is their relatively low replication fidelity with mutation frequencies of 10 Ϫ4 -10 Ϫ5 that result in a heterogeneous virus population often referred to as a quasispecies (7,8). The population consensus sequence defines a particular virus and strain, but closer inspection of individual genomes reveals that they each contain a few random point mutations relative to the consensus. This poo...

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mentioning

confidence: 89%

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

McDonald

Block

Beaucourt

et al. 2016

Journal of Biological Chemistry

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“…Picornaviruses replicate their genome using an RdRp, called 3D pol . 78 Replication takes place in one of two forms: primer-dependent format or de novo RNA synthesis. 79 De novo RNA synthesis, which uses a single initiation nucleotide, gives the 3 0 -hydroxyl group for adding the sequential nucleotide whereas a primer-dependent format uses a protein-based primer or an oligonucleotide for the hydroxyl group donor.…”

Section: The Polymerase Of Rhinovirusesmentioning

confidence: 99%

“…80,81 The 3D pol uses a small viral peptide (VPg) to initiate both plus and minus RNA synthesis in vivo, making Picornaviridae unique in their initiation mechanism. 78 RNA synthesis is initiated using a highly conserved tyrosine residue within VPg using cis-acting replication element as a template whose position varies depending on the genus. 78,82 The 3D pol aids in the binding of two UMP molecules to the tyrosine hydroxyl group of VPg.…”

Section: The Polymerase Of Rhinovirusesmentioning

confidence: 99%

“…78 RNA synthesis is initiated using a highly conserved tyrosine residue within VPg using cis-acting replication element as a template whose position varies depending on the genus. 78,82 The 3D pol aids in the binding of two UMP molecules to the tyrosine hydroxyl group of VPg. [83][84][85] The product of this reaction, VPg-pU is then extended by an additional uridine to form VPg-pUpU.…”

Section: The Polymerase Of Rhinovirusesmentioning

confidence: 99%

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Nucleosides for the treatment of respiratory RNA virus infections

Jordan

Stevens

Deval

2018

Antivir Chem Chemother

125

105

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Influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. In this review, we describe the current medical need resulting from respiratory infections caused by RNA viruses, which justifies drug discovery efforts to identify new therapeutic agents. The RNA polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of RNA chain synthesis. Here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of RNA respiratory viruses: Orthomyxoviridae, Pneumoviridae/Paramyxoviridae, Coronaviridae, and Picornaviridae. We summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. This includes molecules with very broad antiviral spectrum such as ribavirin and T-705 (favipiravir), and others targeting more specifically one or a few virus families. Recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs.

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“…The fingers form a channel that allows entry of the template RNA and ribonucleotide triphosphates (rNTPs) and assist in proper positioning of incoming nucleotides in the active site (21). The palm contains the active site, and the thumb functions in contacting exiting nascent RNA (21)(22)(23). However, there is diversity in the viral genes that encode RdRps; additional domains that perform a variety of functions, such as methyltransferase, endonuclease, polyribonucleotidyl transferase, guanylyltransferase, membrane targeting, protein-protein binding, or protein-RNA binding activities, are often present (24)(25)(26).…”

mentioning

confidence: 99%

Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

Sexton

Smith

Blanc

et al. 2016

J Virol

169

177

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Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. RNA virus replication results in the incorporation of a relatively high number of mutations, ranging from 10 Ϫ4 to 10 Ϫ6 mutations per site per round of replication (1-5). It is thought that low-fidelity replication is largely responsible for the capacity of RNA viruses to evolve rapidly and adapt to new host species and ever-changing environmental pressures (6-8). RNA-dependent RNA polymerase (RdRp) is central to the replication of RNA viruses and is a key regulator of nucleotide selectivity and fidelity (9, 10). Recent studies of coxsackievirus virus B3 (CVB3), poliovirus, HIV-1, and other viruses have demonstrated that viable viruses are recoverable only within a 4-fold range of RdRp fidelity (11)(12)(13)(14). In most cases, altered RdRp fidelity decreases fitness relative to wild-type (WT) viruses; this has been demonstrated for changes as small as a 1.2-fold difference in the accumulation of mutations (12,(14)(15)(16). Despite having as little as no amino acid identity outside conserved motifs (11)(12)(13)(14)(17)(18)(19), all described polymerase structures (including RdRps) resemble a "cupped right hand," with finger, palm, and thumb domains (20). The fingers form a channel that allows entry of the template RNA and ribonucleotide triphosphates (rNTPs) and assist in proper positioning of incoming nucleotides in the active site (21). The palm contains the active site, and the thumb functions in contacting exiting nascent RNA (21-23). However, there is diversity in the viral genes that encode RdRps; additional domains that perform a variety of functions, such as methyltransferase, endonuclease, polyribonucleotidyl transferase, guanylyltransferase, membrane targeting, protein-

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RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms

Cited by 58 publications

References 91 publications

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

Nucleosides for the treatment of respiratory RNA virus infections

Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

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