Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulencein Mice

Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

Jesus

Costa

Gonçalves

et al. 2009

“…The cell and virus culture methods used in this study have been described elsewhere (1). Wild-type VAC and its CDV r derivatives were obtained as described previously (1) and are derived from a stock of VAC (strain WR) originally acquired from the American Type Culture Collection. Viruses carrying temperature-sensitive mutations in the E9L gene (Dts83 and Cts42) were obtained from R. Condit (Gainesville, FL).…”

Section: Methodsmentioning

confidence: 99%

“…However, we have developed a novel, antiviral-based strategy for the study of the role of E9 in viral recombination. This approach was developed from our previous studies of the mechanism of action of the dCMP analog cidofovir {(S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl) cytosine]} (CDV) (34,35) as well as through the characterization of CDV-resistant (CDV r ) VAC strains (1). We have previously shown that VAC DNA polymerase can use the diphosphoryl metabolite of CDV (CDVpp) as a substrate and faithfully incorporate a CDV residue opposite dGMP in the template strand (35).…”

mentioning

confidence: 99%

The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination

Gammon

Evans

2009

Self Cite

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3-to-5 proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3 ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV r ) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3-to-5 exonuclease domain of the viral polymerase. Viruses encoding a CDV r mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3 ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3-to-5 exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

“…Compounds that show promise in preclinical or clinical settings are the cidofovir derivative CMX001 (24), which is a nucleoside analog, and ST-246 (12,13), which has a unique mechanism of action in preventing viral egress from infected cells. With the majority of inhibitors, including CMX001 and ST-246, viral resistance is achieved rapidly in cell culture (1,9,33). As with many antimicrobial strategies, effective treatment is likely to require combination therapy.…”

mentioning

confidence: 99%

Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-Oriented Synthesis Library

Dower

Filone

Hodges

et al. 2012

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drugresistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.