Amplification of the ribonucleotide reductase small subunit gene: analysis of novel joints and the mechanism of gene duplication in vaccinia virus

Slabaugh, Mary B.; Roseman, N A; Mathews, Christopher K.

doi:10.1093/nar/17.17.7073

Cited by 35 publications

(36 citation statements)

References 36 publications

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“…The pool of potential new pathogens is large, so defining biomolecular signatures that may be predictive of agents poised to cross into new species could improve our ability to anticipate and avert epidemics. Gene amplification may be one such marker of imminent adaptation: it is a broad evolutionary mechanism that has been described across all domains of life, including viruses (15,(20)(21)(22)40). In general, gene amplification provides two potential benefits.…”

Section: Discussionmentioning

confidence: 99%

“…The precise mechanism of PKR antagonism by RhTRS1 is still unclear; however, it is known to inhibit the PKR pathway at a step after PKR autophosphorylation (14), and this phenotype was maintained in virus populations containing rhtrs1 amplifications (15). Gene amplification is a universal mechanism of rapid adaptation in eukaryotes (16,17), prokaryotes (18,19), and viruses (15,(20)(21)(22), enabling diverse adaptations such as neofunctionalization, antibiotic resistance, and evasion of host restriction factors (reviewed in reference 23). In general, amplification of a gene with weak activity can increase the fitness of an organism through overexpression related to gene dosage effects.…”

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confidence: 99%

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Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

Brennan

Kitzman

Shendure

et al. 2015

J Virol

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Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it is not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus, rhtrs1, improved replication of a recombinant poxvirus (VV⌬E⌬K؉RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations that improve virus replication with only a single copy of rhtrs1 to evolve, we passaged rhtrs1-amplified viruses in semipermissive primate cells. After passage, we isolated two viruses that contained only a single copy of rhtrs1 yet replicated as well as the amplified virus. Surprisingly, rhtrs1 was not mutated in these viruses; instead, we identified mutations in two vaccinia virus (VACV) genes, A24R and A35R, either of which was sufficient to improve VV⌬E⌬K؉RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the antipoxviral drug isatin-␤-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a "molecular foothold," broadly improving replication to facilitate rapid adaptation, while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification. IMPORTANCEUnderstanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of rhtrs1, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of rhtrs1-amplified viruses, there arose in two vaccinia virus genes mutations that improved viral replication without requiring rhtrs1 amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host. Of the approximately 1,400 known human pathogens, over 60% are zoonotic (1). Furthermore, during the last 80 years, more than 70% of all new and emerging human infectious diseases were acquired from animal sources (2). These emerging zoonoses are often responsible for severe disease in humans, such as HIV-1 infection/AIDS (3) and the recent Ebola pandemic in West Africa (4). Similarly, pathogen transmission between both wild and domes...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

Brennan

Kitzman

Shendure

et al. 2015

J Virol

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“…sistance (mdr) genes in Plasmodium falciparum [5], whereas, in vaccinia virus, duplicated ribonucleotide reductase genes occurred as both direct and inverted repeats [6]. Sequencing of two joints linking units of amplified DNA containing CAD (the enzyme complex carbamyl phosphate synthetase-aspartate transcarbamylase-dihydro-orotase) genes in Drosophila cells gave rise to the hypothesis that in this case two illegitimate recombinations between sister chromatids led to the novel joints and then the amplifications occurred by a series of homologous or further illegitimate recombinations [7].…”

Section: Introductionmentioning

confidence: 99%

Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer)

Field

Devonshire

1997

Biochemical Journal

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Insecticide resistance in the aphid Myzus persicae results primarily from the amplification of genes encoding the insecticidedetoxifying esterase, E4. Here we report the analysis of flanking DNA co-amplified with the E4 gene. The 5h end of this gene has an untranslated leader sequence interspersed by two introns, and the promoter region lacks TATA and CAAT boxes. The DNA breakpoint involved in the generation of the amplification is just upstream (approx. 250 bp) of the putative E4 transcription start site ; thus the E4 gene is very close to the 5h end of the approx.

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“…Further studies demonstrated that duplication of A17 was sufficient to confer a rifampin resistance phenotype. Duplication of other VACV or recombinant genes has been shown to provide resistance to hydroxyurea (27) and allow adaptation to the antiviral action of protein kinase R (28,29). Thus, gene duplication may be a general mechanism for poxviruses and other viruses to overcome environmental stress.…”

mentioning

confidence: 99%

Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance

Erlandson

Cotter

Charity

et al. 2014

J Virol

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Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCEThe present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein for assembly of virus particles. Resistance to rifampin was linked to a partial duplication of the gene encoding the A17 protein, similar to the resistance to hydroxyurea enabled by duplication of the gene encoding the small subunit of ribonucleotide reductase and of the K3L gene to allow adaptation to the antiviral action of protein kinase R. Gene duplication may provide a way for poxviruses and other DNA viruses with high-fidelity DNA polymerases to adjust rapidly to changes in the environment. Poxviruses are large, enveloped, double-stranded DNA viruses that replicate within the cytoplasm (1). Vaccinia virus (VACV), the model poxvirus, is being employed to construct recombinant vaccines, study antiviral mechanisms, and investigate the basic biology of poxviruses, including assembly. Assembly of VACV begins as viral membranes form and curve into a crescent shape, which is stabilized by a honeycomb lattice of capsid-like D13 protein trimers (2-4, 40). The crescents enlarge to form spheres, termed immature virions (IVs) that, upon processing of the A17 mem...

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Amplification of the ribonucleotide reductase small subunit gene: analysis of novel joints and the mechanism of gene duplication in vaccinia virus

Cited by 35 publications

References 36 publications

Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification

Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer)

Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance

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