Human JC virus nuclear factor 1 binding motifs and large tumor antigen region rquired for transactivation of late promoter

Abstract

The nuclear factor 1 (NF-1) motifs, NF-1 II/III, In the two 98-bp repeats of the transcription-regulatory region of JC virus (JCV), have a critical role in brain-specific transcription from the JCV early promoter-enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter-enhancer (JCVL) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in vivo assays, the two wild-type NF-1 II/III sites, but not the third site, were found to be essential for the transactivation of JCVL by JCV T-Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T-Ag antibody. In electrophoretic mobility shift assays, expression of JCV T-Ag increased the binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 11/111 motifs showed that the increased binding specifically required the wild-type NF-1 II/III sequences and confirmed the requirement of T-Ag. To determine the region of T-Ag necessary for transactivation Of JCVL, the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient transactivation. These results indicated that transactivation of JCVL and increased binding require a factor(s) found specifically in glial cells, the JCV NF-1 II/III sites, and the T-Ag amino-terminal region.