No abstract

This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

opep-2 is an Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) early gene in the ie1-ie2 gene region for which there is no homolog in either the archetype virus, Autographa californica MNPV, or Bombyx mori NPV. opep-2 is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early gene ie1, which produces spliced mRNAs. However, distinct from ie1, the multiple mRNAs of opep-2 are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8-12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene an opep-2 deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells.

No abstract