A human cytomegalovirus early promoter with upstream negative and positive cis-acting elements: IE2 negates the effect of the negative element, and NF-Y binds to the positive element.
The human cytomegalovirus early promoter for the UL4 gene, which codes for an early viral envelope glycoprotein designated gpUL4, requires immediate-early viral protein two (IE2) synthesis to be activated (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281, 1989). We investigated the cis-acting and trans-acting factors that regulate transcription from this UL4 promoter. In transient transfection assays, the viral IE2 protein negated the effect of an upstream cis-acting negative element and enhanced downstream gene expression. A cis-acting positive element contributed to the activity of the viral promoter when an upstream cis-acting negative element was deleted or when the viral IE2 protein was present. The cellular protein(s) that binds to the cis-acting negative element requires further investigation. The cellular protein that binds to the cis-acting positive element was characterized. Two DNA sequence-specific protein complexes were detected with DNA probes spanning the region containing the cis-acting positive element and human cytomegalovirus-infected human fibroblast cell nuclear extracts. The more slowly migrating complex was labeled complex A, and the faster was labeled complex B. Only complex B was detected with mock-infected cell nuclear extracts. Competition experiments confirmed the specificity of the A and B complexes. The protein bound to the DNA in both the complexes contacts a CCAAT box imperfect dyad symmetry (5'CCAATCACTGG3'). Either CCAAT box within the dyad symmetry could compete for binding the nuclear factor. Mutation of the CCAAT box dyad symmetry resulted in a decrease of the transcriptional activity from the UL4 promoter. A cellular transcription factor, antigenically related to nuclear factor-Y (NF-Y), was found in both complexes A and B. Events associated with viral infection caused phosphorylation of protein complex A. Dephosphorylation of the DNA-binding protein converts complex A to complex B. The effect of phosphorylation of NF-Y is not known.
Huang
L
L
Department of Microbiology, School of Medicine, University of Iowa, Iowa City 52242.
Malone
C L
CL
Stinski
M F
MF
D000135:Acid Phosphatase / Q000378:metabolism; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D003587:Cytomegalovirus / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism; D014759:Viral Envelope Proteins; D014764:Viral Proteins
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D015534:Trans-Activators; D014157:Transcription Factors; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; D000135:Acid Phosphatase