High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene.

Abstract

Spheroidin (SPH) is the most highly expressed gene of the entomopoxvirus isolated from Amsacta moorei (AmEPV). The level of expression of poxvirus genes is believed to be governed in large part by the promoter. Poxvirus promoters generally consist of approximately 40 bp which frequently terminate at the 3' end with a translation initiating TAAATG sequence. We have examined the requirements for high levels of SPH gene expression by constructing AmEPV recombinants containing either the SPH promoter or the late vertebrate poxvirus promoter derived from the cowpox virus A-type inclusion (ATI) gene. In addition, we have examined SPH promoter derivatives which extend beyond the 3' TAAATG to include 2 or 20 bp of the 5' coding sequence of the SPH gene. Examination of insect cells infected with these AmEPV ATI-lacZ or SPH-lacZ recombinants suggests that ATI-lacZ expression begins 12 h before and is essentially complete prior to any SPH-lacZ expression, allowing functional distinction between the ATI and SPH promoters and implying that different factors regulate the two promoters within the insect environment. SPH promoter-regulated expression is significantly enhanced within infected insect cells by including the additional 20 bp of the N-terminal SPH coding sequences as part of the promoter. However, when any of the SPH promoter constructs, including those containing the downstream sequences, were inserted into vaccinia virus, only very low levels of beta-galactosidase expression were observed. These results imply that downstream coding sequences within the SPH gene enhance SPH gene expression only within the insect environment.