A MECHANISM FOR NEGATIVE GENE-REGULATION IN AUTOGRAPHA-CALIFORNICA MULTINUCLEOCAPSID NUCLEAR POLYHEDROSIS-VIRUS

The Autographa California multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-l) is thought to play a central role i n stimulating early viral transcription, IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulat e the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2, O ur results indicate that IE-1 negatively regulates the expression of c ertain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') nea r their mRNA start sites. The IE-1 binding motif was also found sia-hi n the palindromic sequences of AcMNPV homologous repeat (hr) regions t hat have been shown to bind IE-1, The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by intr oducing mutations in these promoters in which the central 6 bp were re placed with BglII sites, GUS reporter constructs containing ie-2 and p e-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containi ng plasmid (pIe-1). Comparisons of GUS expression produced by the muta nt and wild-type constructs demonstrated that the IE-1 binding motif m ediated a significant decrease in expression from the ie-2 and pe-38 p romoters in response to increasing pIe-1 concentrations, Electrophoret ic mobility shift assays width pIe-1-transfected cell extracts and sup ershift assays with IE-1-Specific antiserum demonstrated that IE-1 bin ds to promoter fragments containing the IE-1 binding motif but does no t bind to promoter fragments lacking this motif.