Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

Liu, Xinran; Musser, Derek M.; Lee, Cheri A.; Yang, Xiaorong; Arnold, Jamie J.; Cameron, Craig Ε.; Boehr, David D.

doi:10.3390/v7102894

Cited by 6 publications

(7 citation statements)

References 53 publications

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“…The discovery of multiple poxviruses in bats leads to inquiries about the possibility of coevolution between the host and virus [50]. Bats, whose species have diverged 50 million years ago, include diverse species in the suborders of “megabats” and “microbats,” differing both on the behavioral and molecular levels [14, 15].…”

Section: Discussionmentioning

confidence: 99%

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat

Nakazawa

Gao

et al. 2017

Virus Genes

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The genome of Eptesipoxvirus (EPTV) is the first poxvirus genome isolated from a microbat. The 176,688 nt sequence, which is believed to encompass the complete coding region of the virus, is 67% A+T and is predicted to encode 191 genes. 11 of these genes have no counterpart in GenBank and are therefore unique to EPTV. The presence of a distantly related ortholog of Vaccinia virus F5L in EPTV uncovered a link with fragmented F5L orthologs in Molluscum contagiosum virus/squirrelpox and clade II viruses. Consistent with the unique position of EPTV approximately mid-point between the orthopoxviruses and the clade II viruses, EPTV has 11 genes that are specific to the orthopoxviruses and 13 genes that are typical, if not exclusive, to the clade II poxviruses. This mosaic nature of EPTV blurs the distinction between the old description of the orthopoxvirus and clade II groups. Genome annotation and characterization failed to find any common virulence genes shared with the other poxvirus isolated from bat (pteropoxvirus); however, EPTV encodes 3 genes that may have been transferred to or from deerpox and squirrelpox viruses; 2 of these, a putative endothelin-like protein and a MHC class I-like protein are likely to have immunomodulatory roles.

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Section: Discussionmentioning

confidence: 99%

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat

Nakazawa

Gao

et al. 2017

Virus Genes

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“…2). Previous kinetics studies had indicated that incorporation of 2Ј-C-Me-AMP occurs with reduced catalytic efficiency (33), as evidenced by a decreased second-order rate constant k pol /K d,app , where k pol is the maximum polymerase rate constant and K d,app is the apparent dissociation constant for the incoming NTP (Fig. 2).…”

Section: Nmr Studies Of 2-c-methylated Nucleotidementioning

confidence: 93%

“…under the time necessary for the NMR experiments). D, the secondorder rate constants for single-nucleotide incorporation based on the ssAU RNA as determined previously (33,51). The kinetic parameters k pol and K d are the maximum polymerase rate constant and the apparent dissociation constant for the incoming NTP, respectively.…”

Section: C-met Probes Report On Ligand Binding and Conformational Changes Throughout The Rdrp Structurementioning

confidence: 99%

2′-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase

Boehr

Arnold

et al. 2019

Journal of Biological Chemistry

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Edited by Wolfgang PetiThe 2-C-methyl ribonucleosides are nucleoside analogs representing an important class of antiviral agents, especially against positive-strand RNA viruses. Their value is highlighted by the highly successful anti-hepatitis C drug sofosbuvir. When appropriately phosphorylated, these nucleotides are successfully incorporated into RNA by the virally encoded RNA-dependent RNA polymerase (RdRp). This activity prevents further RNA extension, but the mechanism is poorly characterized. Previously, we had identified NMR signatures characteristic of formation of RdRp-RNA binary and RdRp-RNA-NTP ternary complexes for the poliovirus RdRp, including an open-to-closed conformational change necessary to prepare the active site for catalysis of phosphoryl transfer. Here we used these observations as a framework for interpreting the effects of 2-C-methyl adenosine analogs on RNA chain extension in solution-state NMR spectroscopy experiments, enabling us to gain additional mechanistic insights into 2-C-methyl ribonucleoside-mediated RNA chain termination. Contrary to what has been proposed previously, poliovirus RdRp that was bound to RNA with an incorporated 2-C-methyl nucleotide could still bind to the next incoming NTP. Our results also indicated that incorporation of the 2-C-methyl nucleotide does not disrupt RdRp-RNA interactions and does not prevent translocation. Instead, incorporation of the 2-C-methyl nucleotide blocked closure of the RdRp active site upon binding of the next correct incoming NTP, which prevented further nucleotide addition. We propose that other nucleotide analogs that act as nonobligate chain terminators may operate through a similar mechanism.

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“…in the absence of other Sabin I mutations) also leads to some attenuation of the encoding PV strain (18). In the presence of other Sabin I substitutions, these effects are largely mitigated (46). Nonetheless, the T362I variant can serve as an important tool in understanding how the conformational dynamics of motif D are (un)related to the realignment of the triphosphate.…”

Section: Metal and Solvent Effects Help To Gauge The Effects Of Amino Acid Substitutions On The Prechemistry Conformational Change And Chmentioning

confidence: 99%

Triphosphate Re‐orientation of the Incoming Nucleotide as A Fidelity Checkpoint in Viral RNA‐dependent RNA Polymerases

et al. 2015

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The fidelity of the virus‐encoded RNA‐dependent RNA polymerase (RdRp) plays an essential role in generating sufficient genetic diversity to escape the bottlenecks of the host's anti‐viral response. For many nucleic acid polymerases, a pre‐chemistry conformational change is at least partially rate‐limiting and represents a key fidelity checkpoint. There has been disagreement in the polymerase field about the structural rearrangements important in this step. One suggestion has been that this step represents a rearrangement of the triphosphate of the incoming nucleotide that will facilitate nucleophilic attack by the primer 3′‐hydroxyl group. We have now gained insight into the triphosphate re‐orientation in the poliovirus (PV) RdRp via 31P NMR. Modeling studies suggest that Lys167 and Arg174 on structural motif F make important interactions with the triphosphate. 31P NMR results demonstrate that WT RdRp interacts with “correct” nucleotide in a way different from “incorrect” nucleotide; R174K RdRp is unable to rearrange correct nucleotide like WT enzyme and instead interacts with correct nucleotide in a similar fashion as incorrect nucleotide; The R174K substitution also substantially reduces enzyme efficiency and leads to very high fidelity. The K167R variant has similar catalytic efficiency as WT when correct nucleotide is incorporated, however, incorrect nucleotide is more readily incorporated. Altogether these results suggest that triphosphate re‐orientation is an essential fidelity checkpoint and interactions with motif F residues are important for this alignment.The research is supported from Penn State start‐up funds and NIH R01 AI104878 to DDB.

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Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

Cited by 6 publications

References 53 publications

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat

2′-C-methylated nucleotides terminate virus RNA synthesis by preventing active site closure of the viral RNA-dependent RNA polymerase

Triphosphate Re‐orientation of the Incoming Nucleotide as A Fidelity Checkpoint in Viral RNA‐dependent RNA Polymerases

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