Proteins of the ERV1/ALR family are encoded by all eukaryotes and cytoplasmic DNA viruses for which substantial sequence information is available. Nevertheless, the roles of these
proteins are imprecisely known. Multiple alignments of ERV1/ALR
proteins indicated an invariant C-X-X-C motif, but no similarity to the
thioredoxin fold was revealed by secondary structure predictions. We chose a virus model to investigate the role of these
proteins as
thiol oxidoreductases. When cells were infected with a mutant vaccinia virus 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
protein, was mutated to
serine. These two cysteines were
disulfide bonded during 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 uninfected cells, suggesting a requirement for an upstream viral
thiol oxidoreductase. Remarkably, the
cysteine-containing domains of the E10R and L1R
viral membrane proteins and the
glutaredoxin are in the cytoplasm, in which assembly of
vaccinia virions occurs, rather than in the oxidizing environment of the endoplasmic reticulum. These data indicated a viral pathway of
disulfide bond formation in which the E10R
protein has a central role. By extension, the ERV1/ALR family may represent a ubiquitous class of cellular
thiol oxidoreductases that interact with
glutaredoxins or
thioredoxins.