REPRESSION OF THE A8L GENE, ENCODING THE EARLY TRANSCRIPTION FACTOR 82-KILODALTON SUBUNIT, INHIBITS MORPHOGENESIS OF VACCINIA VIRIONS

The vaccinia virus early transcription factor (VETF) is a DNA binding protein comprised of 70- and 82-kDa subunits encoded by the D6R and AS L genes, respectively. A previous investigation suggested a novel role for the 70-kDa subunit in the morphogenesis of vaccinia virus particl es. The principal objectives of the present study were to determine if the 82-kDa subunit of VETF is also required for morphogenesis and, if so, whether the block occurs before or after the incorporation of the genome into the assembling virus particle. To address these and other questions, we constructed and characterized a conditionally lethal re combinant vaccinia virus in which the A8L gene is stringently represse d by the Escherichia coli [ac operator system. The amount of 82-kDa pr otein synthesized could be regulated by the amount of inducer: from un detectable to higher than normal levels. Virus replication, as determi ned by plaque formation or virus yield upon synchronous infection, was dependent on inducer. Nevertheless, de novo synthesis of the 82-kDa s ubunit was not required for viral early, intermediate, and late gene e xpression or DNA replication. Overexpression of the ASL gene alone, pr oduced by high concentrations of inducer, inhibited viral late protein synthesis, whereas overexpression of the D6R gene alone or both VETF genes simultaneously had little inhibitory effect. Laser confocal fluo rescence and quantitative electron microscopic analyses revealed that immature and DNA-containing intermediate stage particles accumulated i n the absence of inducer, indicating that the A8L protein has a role i n morphogenesis of the core and subsequent events.