Binding of the human cytomegalovirus 80-kDa immediate-early protein (IE2) to minor groove A/T-rich sequences bounded by CG dinucleotides is regulated by protein oligomerization and phosphorylation.
The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream viral promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as the cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphylococcal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579) bound specifically to a [32P]-labeled CRS oligonucleotide probe in an in vitro electrophoretic mobility shift assay (EMSA). In contrast, no direct interaction with the CRS probes could be detected with IE2 wild-type protein in extracts from infected or transfected mammalian cells or when synthesized by in vitro translation. However, in vitro phosphorylation of GST/IE2(346-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding activity. This process required ATP hydrolysis and could be reversed by subsequent incubation with bacterial alkaline phosphatase. Importantly, dephosphorylation of the constitutively expressed native IE2 protein present in a nuclear extract from the U373(A45) cell line unmasked a specific CRS DNA-binding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound structures of intermediate mobility were formed in EMSA assays when a mixture of staphylococcal Protein A/IE2 and GST/IE2 was coincubated with the CRS probe. Coincubation with a DNA-binding negative but dimerization-competent GST/IE2 deletion mutant competitively inhibited DNA-binding by staphylococcal Protein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacked the ability to both dimerize and bind to DNA failed to influence the mobility of the DNA-bound staphylococcal Protein A/IE2 protein. Therefore, IE2 appears to bind to DNA as a higher-order oligomer in which the presence of subunits with mutant DNA-binding domains interferes with the overall DNA-binding function. A series of point mutations introduced into each of nine conserved motifs throughout the DNA-binding and dimerization domain, all of which abolish the ability of the transfected intact IE2 protein to autoregulate the MIE promoter, also all lacked the ability to bind to CRS sequences as GST/IE2(346-379) fusion proteins. Detailed analysis of point mutations in the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relatively sequence-independent manner to 10-bp-long A/T-rich DNA elements bounded on each side by CG dinucleotides. Moreover, the A/T-rich minor groove binding agent distamycin, but not the G/C-rich minor groove binding agent chromomycin-A3, actively competed with IE2 for binding to the CRS motif in a dose-dependent fashion. In conclusion, IE2 binds preferentially as multimerized dimers to A/T-rich sequences in the minor groove that are flanked on both sides by appropriately spaced CG dinucleotides, and inhibition of the DNA-binding or oligomerization activity by PKA phosphorylation probably accounts for the inactivity of the mammalian and in vitro translated forms of the protein.
Waheed
I
I
Molecular Virology Laboratories, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Chiou
C J
CJ
Ahn
J H
JH
Hayward
G S
GS
D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000737:chemistry; D019281:Dimerization; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D008562:Membrane Glycoproteins; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011061:Poly A / Q000378:metabolism*; D011071:Poly T / Q000378:metabolism*; D011487:Protein Conformation; D015534:Trans-Activators / Q000378:metabolism*; D014709:Vero Cells; D014759:Viral Envelope Proteins; D014764:Viral Proteins
D000596:Amino Acids; D017868:Cyclic AMP-Dependent Protein Kinases; D004247:DNA; D004279:DNA, Viral; D015226:Dinucleoside Phosphates; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D010744:Phosphoric Monoester Hydrolases; D011061:Poly A; D011071:Poly T; D015534:Trans-Activators; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C069186:IE2 protein, Cytomegalovirus; C077406:UL115 protein, Human herpesvirus 5; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; C015772:CpG dinucleotide; C008950:poly A-T