Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch

Voehler, Markus; Eoff, Robert L.; McDonald, W. Hayes; Guengerich, F. Peter; Stone, Michael P.

doi:10.1074/jbc.m109.012542

Cited by 11 publications

(10 citation statements)

References 77 publications

(87 reference statements)

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“…Moreover, two different structures of Pol X were reported, with the only difference being the presence or not of the disulfide bond between Cys-81 and Cys-86. This cysteine-cysteine bridge has been shown to reduce about 10-fold the fidelity of Pol X in in vitro assays compared with results with the reduced form (55). These results might explain the divergence in the biochemical results previously published by Tsai and coworkers (20,29) and our group.…”

Section: Construction and Analysis Of Replication Of The Deletion Mutcontrasting

confidence: 55%

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

Redrejo-Rodríguez

Rodrı́guez

Suárez

et al. 2013

J Virol

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bThe function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information.

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Section: Construction and Analysis Of Replication Of The Deletion Mutcontrasting

confidence: 55%

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

Redrejo-Rodríguez

Rodrı́guez

Suárez

et al. 2013

J Virol

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“…The importance of reversible disulfide bond formation as a central feature of redox regulation is increasingly recognized (15,(19)(20)(21)(22)(23)(24). We have verified the predicted enhancement in Pol β binding affinity using a fluorescence binding assay and a stabilized analog of oxidized XRCC1-NTD.…”

Section: Discussionmentioning

confidence: 73%

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

Cuneo

London

2010

Proc. Natl. Acad. Sci. U.S.A.

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Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase β (Pol β) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol β have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol β, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.disulfide switch | DNA repair | scaffolding protein

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“…Conflicting data for Pol X fidelity have been reported [37, 41, 43]. It has been speculated that the incongruous reports resulted from enzyme redox state differences between the published studies [44], wherein greater fidelity is exhibited by the reduced form of the enzyme [45]. We speculate realization of the DNA mutator model proposed previously for ASFV would be reflected by greater intra-sample genome-wide variation and elevated genomic polymorphisms between the wild-type isolates studied here.…”

Section: Discussionmentioning

confidence: 99%

Intra-epidemic genome variation in highly pathogenic African swine fever virus (ASFV) from the country of Georgia

Farlow

Donduashvili²,

Kokhreidze³

et al. 2018

Virol J

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BackgroundAfrican swine fever virus (ASFV) causes an acute hemorrhagic infection in suids with a mortality rate of up to 100%. No vaccine is available and the potential for catastrophic disease in Europe remains elevated due to the ongoing ASF epidemic in Russia and Baltic countries. To date, intra-epidemic whole-genome variation for ASFV has not been reported. To provide a more comprehensive baseline for genetic variation early in the ASF outbreak, we sequenced two Georgian ASFV samples, G-2008/1 and G-2008/2, derived from domestic porcine blood collected in 2008.MethodsGenomic DNA was extracted directly from low-volume ASFV PCR-positive porcine blood samples and subjected to next generation sequencing on the Illumina Miseq platform. De novo and mapped sequence assemblies were performed using CLCBio software. Genomic illustrations, sequence alignments and assembly figures were generated using Geneious v10.2.4. Sequence repeat architecture was analyzed using DNASTAR GeneQuest 14.1.0.ResultsThe G-2008/1 and G-2008/2 genomes were distinguished from each other by coding changes in seven genes, including MGF 110-1 L, X69R, MGF 505-10R, EP364R, H233R, E199L, and MGF 360-21R in addition to eight homopolymer tract variations. The 2008/2 genome possessed a novel allele state at a previously undescribed intergenic repeat locus between genes C315R and C147L. The C315R/C147L locus represents the earliest observed variable repeat sequence polymorphism reported among isolates from this epidemic. No sequence variation was observed in conventional ASFV subtyping markers. The two genomes exhibited complete collinearity and identical gene content with the Georgia 2007/1 reference genome. Approximately 56 unique homopolymer A/T-tract variations were identified that were unique to the Georgia 2007/1 genome. In both 2008 genomes, within-sample sequence read heterogeneity was evident at six homopolymeric G/C-tracts confined to the known hypervariable ~ 7 kb region in the left terminal region of the genome.ConclusionsThis is the first intra-epidemic comparative genomic analysis reported for ASFV and provides insight into the intra-epidemic microevolution of ASFV. The genomes reported here, in addition to the G-2007/1 genome, provide an early baseline for future genome-level comparisons and epidemiological tracing efforts.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1099-z) contains supplementary material, which is available to authorized users.

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Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch

Cited by 11 publications

References 77 publications

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

Oxidation state of the XRCC1 N-terminal domain regulates DNA polymerase β binding affinity

Intra-epidemic genome variation in highly pathogenic African swine fever virus (ASFV) from the country of Georgia

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