Insertional inactivation of the large subunit of ribonucleotide reductase encoded by vaccinia virus is associated with reduced virulence in vivo

Abstract

To assess whether a fully functional VV ribonucleotide reductase enzyme is required during both in vitro and in vivo replication of VV, three mutant viruses were constructed by marker transfer techniques: M1λ, an M1 insertion mutant; TK −, an insertion mutant of the W thymidine kinase (tk) gene; and M1λ/TK −, a double mutant. Extracts of cells infected with the M1λ or Miλ/TK − mutant viruses were assayed for ribonucleotide reductase activity and it was found that insertional inactivation of the M 1 gene abolished the induction of viral enzyme activity in W-infected cells. Each of the three mutant viruses replicated to levels comparable to the wild-type (WT) virus in BSC 40 (monkey), growing A549 (human lung carcinoma) cells, and serum-starved A549 cells, indicating that a functional M1 gene was not required for viral replication in tissue culture. In contrast, in vivo studies indicate that the loss of viral ribonucleotide reductase activity leads to a mild attenuation of VV. By the intracranial route of inoculation, approximately 10-fold more of the M1λ recombinant than the WT virus was required to produce the average lethal dose for 50% of the population of injected mice.