Baculovirus gp64 gene expression: negative regulation by a minicistron

Abstract

Small upstream open reading frames (ORFs) or minicistrons located in the 5' leader of eukaryotic mRNAs have been shown to play a role in translational regulation of some eukaryotic genes, particularly mammalian proto-oncogenes. A survey of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus genome suggests that at least 10 transcripts from late genes contain potential minicistrons, and at least three of these minicistrons appear to be conserved in homologous genes of the related Orygia pseudotsugata MNPV. The position of the minicistron from one of these genes, gp64, is also conserved in gp64 genes from several baculoviruses, suggesting a potential regulatory function. To identify the potential role of the gp64 minicistron in regulating translation from gp64 late mRNAs, we generated a series of recombinant viruses containing the gp64 promoter and minicistron in combination with a chloramphenicol acetyltransferase reporter gene (cat) inserted into the polyhedrin locus. We first fused a cat reporter in frame with the minicistron coding region to demonstrate that the minicistron initiator ATG was in a context suitable for translational initiation. In subsequent experiments, a cat reporter was fused in frame to the downstream gp64 ORF, and various constructs containing point mutations that inactivated the minicistron were examined. Translational efficiency in the presence and absence of the minicistron was measured by quantitative analysis of gp64-cat RNA and the GP64-CAT protein. In the absence of a functional minicistron, translational efficiency from the downstream gp64-cat reporter ORF increased. Surprisingly, single-point mutations that inactivated the minicistron initiator ATG also resulted in utilization of an upstream in-frame ATG that is found within the minicistron coding region and that is in a poor translational initiation context. Double-point mutation constructs that inactivated both the minicistron initiator ATG and the upstream in-frame ATG also resulted in increased translational efficiency from the downstream gp64-cat ORF. Thus, the gp64 minicistron serves as a negative regulatory element that decreases translation of the gp64 ORF on late mRNAs.