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 multinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione-S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 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 capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+ caused a reduction in activity similar to that when the domain was eliminated completely.
Department of Microbiology, Oregon State University, Corvallis, Oregon, 97331, USA.
S H
SH
Harwood
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L
Li
P S
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Ho
A K
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Preston
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Rohrmann
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014157:Transcription Factors / Q000235:genetics*; D035165:Transcription Factors, General; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; D014764:Viral Proteins; C018648:transcription factor S-II; D000409:Alanine