Sequential structures provide insights into the fidelity of RNA replication

Ferrer-Orta, Cristina; Arias, Armando; Pérez-Luque, R.; Escarmı́s, Cristina; Domingo, Esteban; Verdaguer, N.

doi:10.1073/pnas.0700518104

Cited by 118 publications

(154 citation statements)

References 37 publications

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“…of the RdRP is nearly identical to the unbound form (9). This observation, in combination with the modes of binding observed for divalent metal cations and NTPs in the FMDV complexes, further suggest that the NV RdRP⅐RNA⅐NTP complex is a closed complex trapped immediately prior to catalysis, whereas the FMDV complexes are open complexes trapped at earlier or later steps of the reaction cycle.…”

Section: Resultsmentioning

confidence: 50%

“…This pattern of hydrogen bonding reveals how these highly conserved residues (equivalent to poliovirus RdRP residues 297, 288, and 238, respectively) distinguish ribonucleotides from 2Ј-deoxyribonucleotides (24 -26). A similar network of hydrogen bonds is seen in one of the reovirus⅐NTP⅐RNA complexes (8), but the more open FMDV complexes or binary poliovirus RdRP⅐NTP complexes do not form the same hydrogen-bonding pattern, suggesting that ribonucleotide selection occurs in the closed complex formed immediately before catalysis (9,27).…”

Section: Resultsmentioning

confidence: 73%

“…Arg residues occupy a similar position in all viral RdRPs with known structures, as well as distantly related enzymes like HIV reverse transcriptase ( Table 2). The cytosine and nitrocytosine bases stack tightly against Arg-182, unlike the looser interactions seen in the more open FMDV complexes (9).…”

Section: Resultsmentioning

confidence: 99%

“…Experimentally determined structural information is clearly needed to corroborate and further elaborate the different states identified from kinetic studies, but trapping these different states has proven to be very challenging in RdRPs and other polymerases. Crystal structures of RdRP⅐RNA⅐NTP complexes from reovirus (8) and foot-andmouth disease virus (FMDV) (9) have revealed important aspects of RNA and NTP binding, but key details of the closed catalytic complex formed in step iii remain poorly determined. The reovirus RdRP⅐RNA⅐GTP initiation complex (Protein Data Bank code 1N1H) contains two divalent metal ions at the active site and the NTP bound near the RNA primer terminus.…”

Section: Norwalk Virus (Nv)mentioning

confidence: 99%

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Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Zamyatkin

Parra

Alonso

et al. 2008

Journal of Biological Chemistry

139

187

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Crystal structures of Norwalk virus polymerase bound to an RNA primer-template duplex and either the natural substrate CTP or the inhibitor 5-nitrocytidine triphosphate have been determined to 1.8 Å resolution. These structures reveal a closed conformation of the polymerase that differs significantly from previously determined open structures of calicivirus and picornavirus polymerases. These closed complexes are trapped immediately prior to the nucleotidyl transfer reaction, with the triphosphate group of the nucleotide bound to two manganese ions at the active site, poised for reaction to the 3-hydroxyl group of the RNA primer. The positioning of the 5-nitrocytidine triphosphate nitro group between the ␣-phosphate and the 3-hydroxyl group of the primer suggests a novel, general approach for the design of antiviral compounds mimicking natural nucleosides and nucleotides. Norwalk virus (NV)4 is the prototype species of the Norovirus genus within the Caliciviridae and is a major cause of gastroenteritis outbreaks in developed countries (1). Unfortunately, effective treatments are not currently available for many important diseases caused by NV and related RNA viruses. The virally encoded RNA-dependent RNA polymerase (RdRP) is the central enzyme required for replication (2) and is one of the key targets for the development of novel antiviral agents. Recently, 5-nitrocytidine triphosphate (NCT) was identified as a potent inhibitor of picornaviral polymerases, and the nucleoside 5-nitrocytidine was found to have low toxicity and significant antiviral activity in a cultured cell viral infection model (3). A structural and mechanistic basis for rationalizing the inhibitory activity of NCT and related inhibitors is currently lacking because of a shortage of high resolution structural information on RdRP replication complexes.Details on the structure and mechanism of viral RdRPs are clearly required to understand the replication of RNA viruses and to develop more effective antiviral agents. Previous structural studies of viral RdRPs from positive strand RNA viruses and double-strand RNA viruses indicate that the general features of RdRP architecture are highly conserved throughout a diverse range of viruses (reviewed in Refs. 2 and 4). The threedimensional arrangement of N-terminal, fingers, palm, and thumb domains, as well as the active site residues in motifs A-F are nearly universally shared (5).The structural conservation seen in RdRPs suggests that the enzymatic mechanism of nucleotidyl transfer is also highly conserved. Studies primarily on poliovirus RdRP have revealed many of the basic features underlying the nucleotidyl transfer reaction in RdRPs (6, 7). These studies and others indicate that RdRPs, like other polynucleotide polymerases, follow a fivestep reaction cycle involving (i) the binding of an NTP complementary to the base of the template to form an initial "open" complex, followed by (ii) a conformational change to the "closed" complex, (iii) nucleotidyl transfer and translocation, (iv) a second confor...

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Section: Resultsmentioning

confidence: 50%

Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Norwalk Virus (Nv)mentioning

confidence: 99%

See 2 more Smart Citations

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Zamyatkin

Parra

Alonso

et al. 2008

Journal of Biological Chemistry

139

187

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“…K276 is located at the top of the middle finger and is near the RNA template entry channel, but it does not directly interact with the RNA in any of the picornaviral 3D pol -RNA complexes whose structures have been solved thus far. The structural orientations of the phylogenetically conserved amino acids implicated in the polyadenylation of viral RNA are similar in the atomic structures of 3D pol of enterovirus 71 (35), a group A enterovirus; 3D pol of coxsackievirus B3 (36,37), a group B enterovirus; 3D pol of poliovirus (13,16), a group C enterovirus; 3D pol of rhinovirus type 16 (38,39), species A; 3D pol of rhinovirus type 14 (39), species B; and foot-and-mouth disease virus 3D pol (40,41). These phylogenetically conserved sequences and structures likely contribute to the reiterative transcription of poly(A) and poly(U) sequences during viral RNA replication in these other picornaviruses as well, thereby regulating the lengths of poly(A) at the 3= end of viral RNA.…”

Section: Discussionmentioning

confidence: 99%

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Kempf

Kelly²,

Springer

et al. 2013

J Virol

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Picornaviruses have 3= polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3D pol ) uses a reiterative transcription mechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3D pol influenced the polyadenylation of virion RNA. We identified nine alanine substitution mutations in 3D pol that resulted in shorter or longer 3= poly(A) tails in virion RNA. These mutations could disrupt structural features of 3D pol required for the recruitment of a cellular poly(A) polymerase; however, the structural orientation of these residues suggests a direct role of 3D pol in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3D pol and a template RNA with a defined poly(U) sequence provided data consistent with a template-dependent reiterative transcription mechanism for polyadenylation. The phylogenetically conserved structural features of 3D pol involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA template and product strands.P icornaviruses, like many other positive-strand RNA viruses, have RNA genomes with variable-length 3= poly(A) tails (1). The poly(A) tails of picornaviruses are important for viability (2), with the length of the poly(A) tails influencing the magnitudes of both viral mRNA translation and RNA replication (3, 4). RNA sequences and structures within the 3= nontranslated region are reported to influence both the length of poly(A) tails in picornaviral RNA (5) and the efficiency of virus replication (6, 7). Viral RNA-dependent RNA polymerases (3D pol s) are predicted to catalyze the polyadenylation of picornaviral RNA in a template-dependent manner during viral RNA replication (8); however, the mechanisms by which RNA genomes are polyadenylated during viral RNA replication remain obscure. In particular, there is little understanding of the mechanisms regulating the length of poly(A) tails synthesized during RNA replication. The RNA-dependent RNA polymerases of negative-strand RNA viruses reiteratively transcribe a small poly(U) sequence within intergenic regions of the viral RNA genome to produce long poly(A) tails on viral mRNAs (9). In a similar manner, the poliovirus RNA-dependent RNA polymerase can reiteratively transcribe poly(A) and poly(U) sequences during viral RNA replication, producing poly(A) and poly(U) sequences longer than those in the respective template RNAs (8).Viral RNA-dependent RNA polymerases are well studied at the structural level (10-12), yet there have been no reports describing the structural features of viral polymerases involved in the polyadenylation of viral RNA. The RNA-dependent RNA polymerase of picornaviruses a...

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Picornaviruses

Rowlands

2015

Encyclopedia of Life Sciences

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The name picornavirus is derived from pico for small and ribonucleic acid (RNA), denoting the chemical nature of the genome. Virions are nonenveloped icosahedral particles approximately 30 nm in diameter. The capsid is constructed from 60 copies of each of four structural proteins, which account for 70% of the particle mass and enclose the single‐strand genome of 7500–8500 nucleotides. The positive‐strand genome acts directly as a messenger RNA to template a single polyprotein product. This is subsequently processed by virally encoded proteases into mature active proteins. Several intermediate products have distinct functions to the final fully processed proteins. Replication of the genome is initiated by an uridylated peptide and proceeds via a negative‐strand intermediate template. Picornaviruses are among the smallest pathogens of vertebrates and are responsible for many important diseases in humans and animals. Key Concepts There are effective vaccines against polio, hepatitis A and foot‐and‐mouth disease viruses. In addition, movement control and slaughter is used to control foot‐and‐mouth disease. There are no licensed drugs for picornavirus infections. Genome sequence comparisons have replaced biological and biophysical comparisons as the bases for classification within the family. Picornavirus particles comprise 60 copies each of four structural proteins, which form a quasi‐T1 icosohedral capsid encasing the single‐strand RNA genome. The picornavirus RNA genome functions as a messenger RNA to template the synthesis of a single polyprotein, which is posttranslationally processed by viral proteases. Picornaviruses bind to cell‐specific surface receptors, and this interaction is an important factor in determining host and tissue specificity of each virus. Picornaviruses cannot gain entry to cells by membrane fusion, as is the case for enveloped viruses, and require special mechanisms to breach cellular membranes and safely deliver the genome into the host cell. Genome replication occurs in association with virus‐modified cellular membranes. Viral RNA templates complementary negative‐strand molecules, which in turn template multiple positive‐strand copies. The synthesis of all RNA molecules is initiated by an uridylated peptide primer. The mechanisms of transmission of infection play key roles in the epidemiology of picornavirus infections. Picornaviruses are responsible for a wide range of clinical diseases resulting from multiple factors such as receptor specificity, tissue‐specific susceptibility, virulence and the mechanisms of transmission.

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Sequential structures provide insights into the fidelity of RNA replication

Cited by 118 publications

References 37 publications

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Structural Insights into Mechanisms of Catalysis and Inhibition in Norwalk Virus Polymerase

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Picornaviruses

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