The Baculovirus Transcriptional Transactivator ie0 Produces Multiple Products by Internal Initiation of Translation

Abstract

Ie0 is the only gene of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) that is known to be spliced. In this study, cDNAs of ie0 were isolated, cloned, and sequenced. It was observed that IE0 contains 35 amino acids (aa) added to the N-terminus of IE1. In addition, it was found that the leader sequence of ie0 contains a 4-aa minicistron. To functionally characterize IE0, ie0 cDNAs were expressed under control of either the ie1 or the ie0 promoter. Unexpectedly, examination of ie0 translation products revealed that the predominant product from ie0 mRNAs was not IE0, but IE1. Mutation analysis showed that IE1 translation was preferentially initiated from either of two AUGs found in the first 15 nucleotides (nt) of the ie1 ORF that are internal to the ie0 ORF. It is unknown whether the internal translation initiation occurs via a leaky scanning mechanism or by an internal ribosomal entry site. Transactivation analysis with constructs that had point mutations in the ie1 AUGs and were translated only as IE0 revealed that OpMNPV IE0 is a 14- to 15-fold stronger transactivator than IE1. IE0 was also shown to be autoregulatory and to transactivate early genes in an enhancer-independent or -dependent manner. These results suggest that differential expression of baculovirus early genes can be obtained by coexpression of IE0 and IE1 in infected cells, which may permit subtle regulation of specific sets of viral genes.

The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells

“…2A): the higher molecular mass band corresponded to the expected size of the full-length CfEcR-AcIE0. The lower molecular mass band agreed with the expected size of proteins being internally translated from the IE1 start codon, as has been previously observed with the native ie0 mRNA (Theilmann et al, 2001). Transactivation analysis demonstrated that CAT expression peaked by 21 h of induction and remained constant up to 37 h of induction (Fig.…”

“…1B). Previous studies showed that translation of AcMNPV and OpMNPV ie0 produces both IE1 and IE0 due to internal initiation of translation within the AAD (Theilmann et al, 2001). However, ie0 constructs that have point mutations in the internal ATGs were shown to produce only IE0.…”

“…The splicing event adds 54 and 35 amino acids to the N-terminal AAD of IE1 in AcMNPV and OpMNPV, respectively, to produce IE0 (Chisholm & Henner, 1988;Kovacs et al, 1991a, b;Theilmann & Stewart, 1991;Theilmann et al, 2001). The data from several groups indicate that the additional residues added at the N terminus of IE0 may confer different transcriptional regulatory properties from IE1 (Kovacs et al, 1991b;Pearson & Rohrmann, 1997;Theilmann et al, 2001). In addition, it has recently been shown that ie0 mRNA has novel translational properties, which favour translation from internal start codons resulting in ie0 producing both IE1 and IE0 (Theilmann et al, 2001).…”

“…The data from several groups indicate that the additional residues added at the N terminus of IE0 may confer different transcriptional regulatory properties from IE1 (Kovacs et al, 1991b;Pearson & Rohrmann, 1997;Theilmann et al, 2001). In addition, it has recently been shown that ie0 mRNA has novel translational properties, which favour translation from internal start codons resulting in ie0 producing both IE1 and IE0 (Theilmann et al, 2001). An ie0 mutant that is translated only as IE0 was shown to transactivate gene expression at a level 14-fold higher than IE1 (Theilmann et al, 2001).…”

“…In addition, it has recently been shown that ie0 mRNA has novel translational properties, which favour translation from internal start codons resulting in ie0 producing both IE1 and IE0 (Theilmann et al, 2001). An ie0 mutant that is translated only as IE0 was shown to transactivate gene expression at a level 14-fold higher than IE1 (Theilmann et al, 2001). This indicates that the additional amino acids at the N terminus of IE0 modify the transcriptional activation of this protein relative to IE1.…”