The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus

Abstract

Enviroxime is an antiviral compound that inhibits the replication of rhinoviruses and enteroviruses. We have explored the mechanism of action of enviroxime by using poliovirus type 1 and human rhinovirus type 14 as model systems. By varying the time of drug addition to virus-infected cells, we determined that enviroxime could be added several hours postinfection without significant loss of inhibition. This suggested that the drug targeted a step involved in RNA replication or protein processing. To identify this target, we mapped 23 independent mutations in mutants that could multiply in the presence of 1 microgram of enviroxime per ml. Each of these mutants contained a single nucleotide substitution that altered one amino acid in the 3A coding region. Using oligonucleotide-directed mutagenesis of cDNA clones, we have confirmed that these single-amino-acid substitutions are sufficient to confer the resistance phenotype. In addition, we conducted two experiments to support the hypothesis that enviroxime inhibits a 3A function. First, we determined by dot blot analysis of RNA from poliovirus-infected cells that enviroxime preferentially inhibits synthesis of the viral plus strand. Second, we demonstrated that enviroxime inhibits the initiation of plus-strand RNA synthesis as measured by the addition of [32P]uridine to 3AB in poliovirus crude replication complexes. To our knowledge, this is the first evidence that 3A can be targeted by antiviral drugs. We anticipate that enviroxime will be a useful tool for investigating the natural function of the 3A protein.