Identification and characterization of gene products of ORF U90/89 of human herpesvirus 6.

Human herpesvirus 6 (HHV-6) isolates can be classified into two variants, A and B. Comparison of genomic sequences of these variants has highlighted sequence variability in the region spanning U86 to U100. This region includes the immediate early A (IE-A) locus that was defined as positional homologue of the major IE locus of Human cytomegalovirus (HCMV) with little recognizable sequence homologies. A 3.5 kb transcript, one of the four spliced transcripts identified in the IE-A locus, is derived from the U90/89 ORF encoding the IE1 protein. We expressed six Escherichia coli fragments spanning the HHV-6A U90/89 ORF as IE1 fusion proteins. The bacterially expressed fusion protein was used to raise monospecific polyclonal antiserum for detection and identification of the IE1 protein product(s). Using this antiserum we detected 165, 190, and > 190 K proteins in HHV-6A- and HHV-6B-infected cells and the 165 K protein in cells transfected with an IE1 cDNA construct. The IE1 proteins exhibited perinuclear and cytoplasmic localization in infected cells. There was a correlation between the expression level of IE1 and the degree of permissiveness for virus growth in various cell lines. In transient expression experiments a 140 bp fragment from the upstream IE-A region was shown to possess promoter activity. The C-terminal region of IE1 delineated by amino acids (aa) 588 to 636 showed a DNA binding activity in Southwestern blot analysis.

The Stat1 binding motif of the interferon-gamma receptor is sufficient to mediate Stat5 activation and its repression by SOCS3.

Signal transduction via the interferon-gamma (IFN-gamma) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-gamma is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-gamma receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-gamma. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-gamma receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-gamma-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Stat-dependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-gamma-responsive genes.