Dual NF1-requiring effect of human neurotropic JC virus composite pentanucleotide repeat elements on early and late viral gene expression

Abstract

Human polyoma JC virus (JCV) is a largely brain cell-specific virus that is associated with AIDS. The neurotropism involves the various transcription factors interacting with their cognate sequences in the JCV early (JCV(E)) and late (JCV(L)) promoters-enhancer, but the mechanism is unclear. The JCV tandem AGGGA pentanucleotide double repeat element (Pnt2) and the abutting NF1 site form a composite Pnt2 (cPnt2). Here, we studied the roles of both sites of cPnt2 in the expression of JCV(E) and JCV(L). JCV(E) activity was progressively increased in U-87 MG human glioblastoma cells following the site-specific mutation of Pnt2 in one and both 98-bp repeats. In contrast, the activity for JCV(L) was progressively reduced by mutating single and double Pnt2s. However, JCV(E) activity was unaffected by mutating both Pnt2s when both NF1 sites of cPnt2 were mutated. The activity of JCV(L) was also unaffected by double Pnt2 mutations after the NF1 sites were mutated. Differentiating P19 glial cells showed similar results. Next, Pnt2-interacting proteins were examined by two in vitro assays with a Pnt2 oligonucleotide. Mobility shift assays showed one Pnt2-protein complex for U-87 MG cell extracts and two for P19 glial cell extracts. However, similar results were obtained for glial, muscle, and undifferentiated P19 cells. In UV cross-linking assays, two Pnt2-binding proteins (Pnt2BPs) were detected. The results suggested that the effects on JCV(E) and JCV(L) expression of Pnt2BP and NF1 via cPnt2 are dual and require a glial cell-specific NF1 function.