The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a c
alicivirus, is known to have a conserved GDD amino acid motif and several a
dditional regions of sequence homology with all types of polymerases. To te
st whether both aspartic acid residues are in fact involved in the catalyti
c activity and metal ion coordination of the enzyme, several defined mutati
ons have been made in order to replace them by glutamate, asparagine, or gl
ycine, All six mutant enzymes were produced in Escherichia coli, and their
in vitro poly(U) polymerase activity was characterized. The results demonst
rated that the first aspartate residue was absolutely required for enzyme f
unction and that some flexibility existed with respect to the second, which
could be replaced by glutamate.