Elucidating the essential role of the A14 phosphoprotein in vaccinia virus morphogenesis: construction and characterization of a tetracycline-inducible recombinant.

We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-beta-D-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of "empty" crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.