Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein

Abstract

Autographa califomica multicapsid nuclear polyhedrosis virus-infected insect cells express a viral immediateearly transcriptional regulatory protein, IEl, that has been shown by transient-expression assays to stimulate the expression of certain baculovirus delayed-early (DE) promoters and to inhibit the expression of other immediate-early (IE) genes. It is believed that certain DE promoters are activated, in part, by direct interactions between IEl and enhancer elements located in regions adjacent to these genes. We have used transient cotransfection and DNA-binding assays to examine the function of mutant forms of IEl. Our results indirectly show that IEl has at least two separable domains that are essential for its role in the modulation of baculovirus gene expression. A domain rich in acidic residues and essential for transactivation is located within the N-terminal 145 amino acids of the polypeptide. A second domain, located in the C-terminal 437 amino acids

DNA-dependent transregulation by IE1 of Autographa californica nuclear polyhedrosis virus: IE1 domains required for transactivation and DNA binding

“…N-terminal IE1 residues 8 to 118 were sufficient for maximal GAL4-IE1 transactivation. These findings confirmed and extend a deletion analysis by Kovacs et al (23), which showed that the N and C termini of IE1 are required for transactivation and hr complex formation, respectively. Thus, to further distinguish IE1 residues required for transactivation and oligomerization-dependent DNA binding, we tested the capacity of an extensive series of IE1 mutations to bind to an hr5 28-mer-containing DNA probe in electrophoretic mobility shift assays (EMSAs).…”

“…Taken together, these data indicated that N-terminal IE1 residues are sufficient for GAL4targeted transactivation. Since IE1 residues 1 to 8 are dispensable for transactivation (23), our data suggest that the IE1 transactivation domain(s) lies between residues 8 and 118.…”

“…Mutations within the hr5 28-mer that disrupt IE1 binding and eliminate 28-mer-mediated transcriptional activation support a model wherein IE1 transactivation involves sequence-specific DNA binding (23,45). To test IE1's capacity to transactivate by direct DNA binding, we constructed GAL4-IE1 hybrids in which the GAL4 DBD (residues 1 to 147) was fused to IE1 ( Fig.…”

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

“…The analysis focused on a virus population with a high number of passages, as we wanted to focus on the dynamics of a population containing DIs rather than their de novo generation, which has already been documented [5,6,24,35]. The concentration of ie1 was used as a proxy for helper virus titers, because this gene encodes an essential transcriptional regulator [36]. All viruses capable of autonomous replication must therefore carry ie1.…”

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

“…All of the core DNA replication factors bind to DNA and the binding by DNApol and P143 is inferred by their functions in DNA synthesis and in strand separation, respectively. IE-1 binds to replication origin DNA sequences called homologous repeats (hrs) (Kovacs et al, 1992;Pearson et al, 1992). LEF-3 binds to single-stranded DNA (Hang et al, 1995) and binds to P143 (Wu and Carstens, 1998) and to IE-1 (Hefferon and Miller, 2002).…”