REGULATED EXPRESSION OF BACTERIOPHAGE RNA POLYMERASES IN MAMMALIAN CELLS INFECTED WITH RECOMBINANT VACCINIA VIRUSES

Abstract

A system for the inducible expression of bacteriophage RNA polymerases by recombinant vaccinia virus (VV) vectors is described. The genes encoding the bacteriophage SP6, T3, or 1'7 RNA polymerases were cloned into VV transfer vector DNA containing genetic elements from the Escherichia coli (E. coli) lac operon. Bacteriophage RNA polymerases were placed under the control of the late VV promoter 4b and either one or two 21-bp synthetic operators derived from the authentic lac operator, and gene cassettes integrated into the VV genome. W recombinants were tested for regulated expression of bacteriophage RNA polymerases by transfection of recombinant virus infected cell monolayers with expression cassettes containing bacteriophage RNA polymerase promoter controlled β-galactosidase or luciferase reporter genes. Expression of the bacteriophage RNA polymerases was induced with isopropyl-β-D-Thiogalactopyranoside (IPTG), as measured by the amount of 13-galactosidase or luciferase activity produced. Regulated expression of these RNA polymerases was found in separate cell monolayers infected with each of five VV recombinants (vSP6-0P1, vSP6-0P2, vT3-0P1, vT7-0P1, and vT7-0P2); the genetic elements of another recombinant virus (vT3-0P2) were found to be non-functional. Experiments characterizing optimum harvest time post infection [hours post infection (hpi)] and optimum IPTG concentration for reporter gene expression showed that vSP6-0P1 and vSP6-0P2 expressed optimal levels of SP6 RNA polymerase at 30 hpi after induction of expression by the addition of 1.25 mM IPTG. However, regulated expression of T7 RNA polymerase by the viruses vT7- 0P1 and vT7-0P2 was not observed at 30 hpi. Utilizing a 12 hpi harvest time, optimal, regulated expression of 17 RNA polymerase in cell monolayers infected by vT7-0P1 and vT7-0P2 was demonstrated after addition of 0.2 mM 1PTG. Expression of T3 RNA polymerase in cells infected with vT3-0P1 and vT3-0P2 was not as completely analyzed due to inconsistencies in the reporter gene cassettes under the control of the 13 promoter. Levels of SP6 RNA polymerase expression in cells infected with vSP6-0P1 or vSP6-0P2 were similar to levels of T7 RNA polymerase expression observed in cells infected with vT7-0P1 or vT7- 0P2 at 12 hpi, utilizing 0.2 mM IPTG to induce expression of bacteriophage polymerases. Maximum bacteriophage RNA polymerase activity, as measured by luciferase activity, was observed in cell monolayers infected with vSP6-0P1 or vSP6-0P2. Greater than 97% repression of bacteriophage RNA polymerase expression was shown in cell monolayers infected with either of the 0P2 constructs in the absence of the inducer IPTG. These VV recombinants are potentially useful in the expression of a variety of heterologous genes, particularly those coding for proteins that are generally toxic to mammalian cells or viral replication.