Predicted Poxvirus Fen1-Like Nuclease Required For Homologous Recombination, Double-Strand Break Repair And Full-Size Genome Formation

Poxviruses encode many if not all of the proteins required for viral genome replication in the cytoplasm of the host cell. In this context, we investigated the function of the vaccinia virus G5 protein because it belongs to the FEN1-like family of nucleases and is conserved in all poxviruses. A vaccinia virus G5 deletion mutant was severely impaired, as the yield of infectious virus was reduced by approximately two orders of magnitude. The mutant virions contained an apparently normal complement of proteins but appeared spherical rather than brick-shaped and contained no detectable DNA. The inability of G5 with substitutions of the predicted catalytic aspartates to complement the deletion mutant suggested that G5 functions as a nuclease during viral DNA replication. Although the amount of viral DNA produced in the absence of G5 was similar to that made by wild-type virus, the mean size was approximately one-fourth of the genome length. Experiments with transfected plasmids showed that G5 was required for double-strand break repair by homologous recombination, suggesting a similar role during vaccinia virus genome replication.