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

Abstract

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...

Poxvirus membrane biogenesis

“…D13 has no transmembrane domain and associates with the viral membrane through interaction with the N-terminus of A17 (Bisht et al, 2009; Unger et al, 2013). This interaction is supported by a recent finding that duplication or over expression of A17 confers resistance to rifampicin (Erlandson et al, 2014). The removal of D13 trimers during the transition from IV to MV is associated with processing of A17 (Bisht et al, 2009).…”

Emergence of a Viral RNA Polymerase Variant during Gene Copy Number Amplification Promotes Rapid Evolution of Vaccinia Virus

“…Interestingly, two of the seven breakpoints identified in this study were identical to those observed in virus populations passaged in HeLa cells (14), suggesting the presence of these variants at a level below the limit of detection in the ΔE3L parent virus or indicating that these are preferential sites for recombination. These results, combined with studies demonstrating CNV in other poxvirus genes in response to selective pressure (24)(25)(26), continue to reveal CNV as a mechanism for rapid adaptation of VACV. a The ORF containing the mutation is shown for variant alleles (bold) not present in the ΔE3L parent virus (also see Fig.…”

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

“…The observation of gene amplification after only a few passages in several independent experiments (15,21,22) suggests that gene amplification may be a frequent occurrence during VACV replication. Our deep-sequencing data revealed rare instances of a specific J2R-L5R breakpoint in PRO1190-adapted viruses that have only a single copy of rhtrs1 ( Fig.…”

“…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.…”

“…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.…”