A viral member of the ERV1/ALR protein family participates in a cytoplasmic pathway of disulfide bond formation

Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasm ic DNA viruses for which substantial sequence information is available. Nev ertheless, the roles of these proteins are imprecisely known. Multiple alig nments of ERV1/ALR proteins indicated an invariant C-X-X-C motif, but no si milarity to the thioredoxin fold was revealed by secondary structure predic tions. We chose a virus model to investigate the role of these proteins as thiol oxidoreductases. When cells were infected with a mutant vaccinia viru s in which the E10R gene encoding an ERV1/ALR family protein was repressed, the disulfide bonds of three other viral proteins-namely, the L1R and F9L proteins and the G4L glutaredoxin-were completely reduced. The same outcome occurred when Cys-43 or Cys-46, the putative redox cysteines of the E10R p rotein, was mutated to serine. These two cysteines were disulfide bonded du ring a normal virus infection but not if the synthesis of other viral late proteins was inhibited or the E10R protein was expressed by itself in uninf ected cells, suggesting a requirement for an upstream viral thiol oxidoredu ctase. Remarkably, the cysteine-containing domains of the E10R and L1R vira l membrane proteins and the glutaredoxin are in the cytoplasm, in which ass embly of vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum, These data indicated a viral pathway of disul fide bond formation in which the E10R protein has a central role. By extens ion, the ERV1/ALR family may represent a ubiquitous class of cellular thiol oxidoreductases that interact with glutaredoxins or thioredoxins.