ACMNPV LATE EXPRESSION FACTOR-5 INTERACTS WITH ITSELF AND CONTAINS A ZINC RIBBON DOMAIN THAT IS REQUIRED FOR MAXIMAL LATE TRANSCRIPTION ACTIVITY AND IS HOMOLOGOUS TO ELONGATION-FACTOR TFIIS

The late expression factor-5 gene (lef-5) of Autographa californica mu ltinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expr ession. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione-S-transferase affin ity assays. Deletion analysis suggested that the C-terminal 71 amino a cids (aa) were not required for interaction. However, all deletions te sted involving the N-terminal 194 aa significantly reduced LEF5:LEF-5 interaction. LEF5 or LEF5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a beta -glucuronidase (GUS) reporter gene under control of the late vp39 caps id promoter. Amino-acid sequence analysis of LEF5 identified a previou sly unreported domain within the C-terminal 32 aa that is homologous t o the zinc ribbon domain of RNA polymerase II elongation factor IIS (T FIIS) from a variety of taxa. Molecular modeling of the putative LEF5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS sugg ested that this domain could fold into a Zn ribbon structure similar t o TFIIS. Alanine scanning mutagenesis of amino acids predicted to be i mportant for functioning of the LEF-5 ribbon structure significantly r educed LEF5 activity in transient expression assays. Mutations changin g the amino acids predicted to coordinate Zn2+ caused a reduction in a ctivity similar to that when the domain was eliminated completely. (C) 1998 Academic Press.