Isolation and characterization of cidofovir resistant vaccinia viruses

Becker, Marie N.; Obraztsova, Maria; Kern, Earl R.; Quenelle, Debra C.; Keith, Kathy A.; Prichard, Mark N.; Luo, Ming; Moyer, Richard W.

doi:10.1186/1743-422x-5-58

Cited by 36 publications

(48 citation statements)

References 20 publications

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“…A recent study by Becker et al has shown that VACVs encoding only the A314V substitution are sevenfold more resistant to HPMPC than wild-type VACV; however, these viruses grow poorly in culture (5), unlike our VACV encoding the A314T substitution, which replicated as well as wild-type virus in culture (2). These results suggest that while both substitutions confer similar levels of resistance, viruses encoding the A314T substitution are likely more fit for replication in culture, although a consistent finding among these studies is that HPMPC R viruses were highly attenuated in their virulence in mouse models (2,5). Our previous study also identified an alanine-to-valine substitution at position 684 in the putative polymerase domain of our HPMPC R VACV DNA polymerase genes (2).…”

Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hycontrasting

confidence: 44%

“…Interestingly, three separate studies found that the passage of VACV in the presence of HPMPC leads to the selection of virus encoding either an alanine-tothreonine (2) or alanine-to-valine substitution at position 314 in the viral DNA polymerase (5,33). Our previous study clearly showed through marker rescue experiments that the A314T substitution could confer an approximately fivefold increase in HPMPC resistance in VACV compared to wild-type virus (2).…”

Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hymentioning

confidence: 97%

“…The finding that the DNA polymerase is the drug target is shown by the fact that exposing VACV to HPMPC in vitro selects for mutations in the E9L (DNA polymerase) gene that likely affect both the 3Ј-to-5Ј exonuclease and the 5Ј-to-3Ј polymerase activities (2,5,33). Interestingly, three separate studies found that the passage of VACV in the presence of HPMPC leads to the selection of virus encoding either an alanine-tothreonine (2) or alanine-to-valine substitution at position 314 in the viral DNA polymerase (5,33).…”

Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hymentioning

confidence: 99%

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Mechanism of Antiviral Drug Resistance of Vaccinia Virus: Identification of Residues in the Viral DNA Polymerase Conferring Differential Resistance to Antipoxvirus Drugs

Gammon

Snoeck²,

Fiten

et al. 2008

J Virol

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The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a ϳ15-foldincreased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

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Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hycontrasting

confidence: 44%

Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hymentioning

confidence: 97%

Section: Times In Medium Containing An Escalating Dose Of (S)-1-[3-hymentioning

confidence: 99%

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Mechanism of Antiviral Drug Resistance of Vaccinia Virus: Identification of Residues in the Viral DNA Polymerase Conferring Differential Resistance to Antipoxvirus Drugs

Gammon

Snoeck²,

Fiten

et al. 2008

J Virol

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“…Several in vitro studies with purified DNA polymerase of human cytomegalovirus and VACV (33,34,55), and in vivo studies with CDV-resistant viruses (2,5,28,45) have pointed to virus DNA replication as the main target for CDV action. However, no detailed analysis of the effect of CDV on the VACV replicative cycle has ever been reported.…”

Section: Discussionmentioning

confidence: 99%

“…Differences between the effects of CDV on human cytomegalovirus (55) and VACV enzymes have been observed, but overall it has been widely accepted that CDV acts by inhibiting the process of virus DNA replication. Moreover, most CDV-resistant VACV strains contain mutations in the catalytic domain or in the 3Ј-5Ј exonuclease domain of the DNA polymerase (2,5,28,45).…”

mentioning

confidence: 99%

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

Jesus

Costa

Gonçalves

et al. 2009

J Virol

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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.

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Detection of Human-Pathogenic Poxviruses

Kurth

Nitsche

2010

Diagnostic Virology Protocols

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With the eradication of smallpox about 30 years ago, the identification and differentiation of other poxviruses with varying pathogenicity in humans present a challenge for diagnostic facilities. While a clinical differentiation can be demanding, electron microscopy is the fastest approach to identify poxviruses. Molecular techniques, based on specific genomic sequences, are routinely applied to identify poxvirus species and distinguish between individual virus variants. In this chapter, we present detailed protocols for both techniques and discuss questions relevant to fast and reliable diagnostics of poxviruses.

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Isolation and characterization of cidofovir resistant vaccinia viruses

Cited by 36 publications

References 20 publications

Mechanism of Antiviral Drug Resistance of Vaccinia Virus: Identification of Residues in the Viral DNA Polymerase Conferring Differential Resistance to Antipoxvirus Drugs

Mechanism of Antiviral Drug Resistance of Vaccinia Virus: Identification of Residues in the Viral DNA Polymerase Conferring Differential Resistance to Antipoxvirus Drugs

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

Detection of Human-Pathogenic Poxviruses

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