Recombinant vaccinia viruses that express the
bacteriophage T3 RNA polymerase (VV-T3pol) or the Escherichia coli
lac repressor (VV-lacI) under control of the early-late
vaccinia promoter P7.5 were constructed. To determine whether phage polymerase and
lac repressor can function in the nucleus of mammalian cells, the bacterial
chloramphenicol acetyltransferase (CAT) gene was cloned downstream of a T3 promoter (PT3-CAT) or downstream of a T3 promoter-lac operator fusion
element (PT3Olac-CAT), and these reporter gene cassettes were introduced stably into NIH 3T3 or Ltk- cells.
Infection of 3T3/PT3-CAT or Ltk-/PT3-CAT cells by VV-T3pol led to rapid expression of CAT (greater than 20 ng of CAT
protein per 10(6) cells). The presence of
hydroxyurea (which blocks virus DNA replication) did not prevent CAT production. When 3T3/PT3Olac-CAT cells were infected with both VV-T3pol and VV-lacI (multiplicities of
infection of 2.5 and 10, respectively), greater than 30-fold repression of CAT gene activity by
lac repressor was observed. This could be reversed to unrepressed levels by the presence of 10 mM o-nitrophenyl-beta-D-galactoside (
IPTG) in the medium. Regulated expression of the target gene was observed with cell lines that had been maintained for over 1 year (greater than 50 passages in culture), and Southern blot analysis revealed the presence of the CAT gene only in the nuclear fraction in these cells, demonstrating the stability of the target gene. These results indicate that vaccinia virus-encoded
proteins can function in the mammalian nucleus and provide the basis for a genetic system in which essential vaccinia virus genes, placed in the chromosome of a cell, can be used to
complement defective virus particles. This approach may prove useful for other virus systems.