Binding of the human cytomegalovirus 80-kDa immediate-early protein (IE2) to minor groove A/T-rich sequences bounded by CG dinucleotides is regulatedby protein oligomerization and phosphorylation

The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream vira l promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as t he cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphyloc occal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579 ) bound specifically to a [P-32]-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 protei n in extracts from infected or transfected mammalian cells or when synthesi zed by in vitro translation. However, in vitro phosphorylation of GST/IE2(3 46-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding ac tivity. This process required ATP hydrolysis and could be reversed by subse quent incubation with bacterial alkaline phosphatase. Importantly, dephosph orylation of the constitutively expressed native IE2 protein present in a n uclear extract from the U373(A45) cell line unmasked a specific CRS DNA-bin ding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound struct ures 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/I E2 deletion mutant competitively inhibited DNA-binding by staphylococcal Pr otein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacke d the ability to bath dimerize and bind to DNA failed to influence the mobi lity 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-bi nding function. A series of point mutations introduced into each of nine co nserved motifs throughout the DNA-binding and dimerization domain, all of w hich abolish the ability of the transfected intact IE2 protein to autoregul ate 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 i n the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relat ively sequence-independent manner to 10-bp-long A/T-rich DNA elements bound ed on each side by CG dinucleotides. Moreover, the A/T-rich minor groove bi nding agent distamycin, but not the G/C-rich minor groove binding agent chr omomycin-A3, actively competed with IE2 for binding to the CRS motif in a d ose-dependent fashion. In conclusion, IE2 binds preferentially as multimeri zed dimers to A/T-rich sequences in the minor groove that are flanked on bo th sides by appropriately spaced CG dinucleotides, and inhibition of the DN A-binding or oligomerization activity by PKA phosphorylation probably accou nts for the inactivity of the mammalian and in vitro translated forms of th e protein. (C) 1998 Academic Press.