Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Arnold, Jamie J.; Ghosh, Saikat; Cameron, Craig Ε.

doi:10.1074/jbc.274.52.37060

Cited by 103 publications

(37 citation statements)

References 42 publications

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“…Interestingly, Mg 2ϩ , the most common divalent metal ion used by many RNA polymerases, cannot support ZIKV NS5 primer extension activity. The effect of Mn 2ϩ preference is not unique to the ZIKV polymerase and has been observed with other viral polymerases, including the NS5 protein of Japanese encephalitis virus (JEV), also a Flavivirus member (32,33). In our study, a high concentration of Mg 2ϩ inhibited ZIKV NS5 polymerase activity even in the presence of Mn 2ϩ , indicating perhaps that Mg 2ϩ can compete with Mn 2ϩ for binding to the active site of the ZIKV NS5.…”

Section: Discussionsupporting

confidence: 53%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

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Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2ϩ is required for enzymatic activity, while Mg 2ϩ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5=-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2=-C-methyl-and 2=-C-ethynyl-substituted analog 5=-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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Section: Discussionsupporting

confidence: 53%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

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“…Picornavirus genomic (plus-strand) and antigenomic (minus-strand) RNAs contain a protein (VPg) linked to the 5Ј-end. Attachment is thought to occur by using uridylylated VPg (VPgpUpU) as primer for the virus-encoded RNA-dependent RNA polymerase (3Dpol), an enzyme that it is quite inefficient at initiation of RNA synthesis de novo (30). VPg-pUpU can be produced by the polymerase by using a genome-specific RNA stem-loop structure (oriI) (3,4) or a poly(rA) tail (5).…”

Section: Discussionmentioning

confidence: 99%

“…The truncations will clearly have a large effect on the stability of the stem. Because Mn 2ϩ binds to the phosphodiester backbone more tightly than Mg 2ϩ , stability of the stem should be enhanced by using Mn 2ϩ as the divalent cation in the reaction (30). The use of Mn 2ϩ causes a 3-fold reduction in the efficiency of uridylylation relative to Mg 2ϩ (compare black bars with gray bars for 61-nt RNA in Fig.…”

Section: A 29-nt Orii Element and Protein 3c Are Sufficient To Directmentioning

confidence: 99%

Picornavirus Genome Replication

Pathak¹,

Arnold

Wiegand

et al. 2007

Journal of Biological Chemistry

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All picornaviruses have a protein, VPg, covalently linked to the 5-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPgpUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication.Picornaviruses are the etiologic agent of numerous diseases of medical and veterinary importance. Poliomyelitis, the common cold, summer flu, hepatitis, and foot-and-mouth disease can all be caused by picornaviruses (1). These viruses have a single-stranded RNA genome of positive polarity that is on the order of 7500 nt 3 in length (1). A protein, VPg (virion protein genome-linked), is covalently linked to the 5Ј-end of the viral genome, the so-called plus-strand, and a poly(rA) tail is present at the 3Ј-end (1). Genome replication occurs in a process that uses the plus-strand as a template for minus-strand synthesis, which, in turn, is used as a template for production of an excess of plus-strands (2). Initiation of both plus-and minus-strand RNA synthesis is thought to be primed by a uridylylated form of VPg, VPg-pUpU (2). Several years ago, Paul and Wimmer made the paradigmshifting observation that an RNA stem-loop structure in the 2C-coding region of the poliovirus (PV) genome was capable of templating production of VPg-pUpU (3, 4) much more efficiently than the poly(rA) tail (5). Since this time, it has become clear that all picornaviruses appear to use a similar strategy for production of VP-pUpU (6 -12). All picornaviruses have a cisacting RNA element capable of templating the production of VPg-pUpU, although the position of this element in the genome varies. This element has been termed oriI...

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“…Previous studies of 3D pol -catalyzed nucleotide incorporation that utilized short, heteropolymeric RNA primer-templates as substrates demonstrated that this enzyme binds to primertemplate such that some enzyme has the 3Ј-OH from the "primer" strand in the catalytic center ("correct" orientation) and some enzyme has the 3Ј-OH from the "template" strand in the catalytic center ("incorrect" orientation) (30). When bound in the "incorrect" orientation, 3D…”

Section: ؊1mentioning

confidence: 99%

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Arnold

Cameron

2000

Journal of Biological Chemistry

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142

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Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D pol , have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as "sym/sub." Formation of 3D pol -sym/sub complexes is slow owing to a slow rate of association (0. pol -sym/sub product complexes have a half-life of 8 h at 22°C. The stability of 3D pol -sym/sub complexes is temperature-dependent. At 30°C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D pol dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D pol and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.Critical to the multiplication of all viruses are transcription and replication of the viral genome. In the case of positivestrand RNA viruses, these processes are catalyzed by the virusencoded RNA-dependent RNA polymerase (RdRP).1 Specific inhibitors of the RdRP should serve as potent antiviral agents.Indeed, inhibitors of viral DNA polymerases and reverse transcriptase (RT) are used clinically to treat infection by herpesviruses and human immunodeficiency virus (1, 2). Of course, cellular polymerases are also potential targets for antiviral chemotherapeutics that inhibit viral polymerases. Therefore, target specificity is of central importance to the development of clinically useful compounds. In some cases, drug activation only occurs in virus-infected cells thereby increasing specificity and diminishing toxicity (1). In others, however, infected cellspecific, activation mechanisms are not an option, and other approaches are required (2).A comprehensive understanding of the differences between viral and cellular polymerases facilitates the design of specific inhibitors. In the case of human immunodeficiency virus RT, a combination of detailed kinetic (3-12) and structural (13-18) information was essential to provide an understanding of the molecular basis for RT function and the mechanism of action of non-nucleoside inhibitors of RT (9, 10). In fact, a description of the nucleotide addition cycle catalyzed by all classes of polymerases studied to date has benefited from both kinetic and structural analyses (19 -28). The "structural era" for the RdRP has begun with the recent solution of the x-ray crystal structure for the RdRP from poliovirus, 3D pol (29). For this reason, poliovirus 3Dpol is the ideal model system to use in the study of RdRP function. Unfortunately, detailed, mechanistic information regarding 3D pol -catalyzed nucleotide incorporation is sparse. The existing gap derives from the inability to assemble stoichiometrically 3D pol -primer-t...

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Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Abstract: . Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D pol -catalyzed nucleotide incorporation.

Cited by 103 publications

References 42 publications

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Picornavirus Genome Replication

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

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