Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b

The 86-kDa IE2 protein (IE2-p86) of human cytomegalovirus (HCMV) is a poten t transactivator of viral as well as cellular promoters, Several lines of e vidence indicate that this broad transactivation spectrum Is mediated by pr otein-protein interactions, To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of 1E2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the is olation of two ubiquitin-homologous proteins. SUMO-1 and hSMT3b, as well as their conjugating activity hUBC9 (human ubiquitin-conjugating enzyme 9) as specific interaction partners of HCMV IE2. The polypeptides SUMO-1 and hSM T3b have previously been shown to be covalently coupled to a subset of nucl ear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By West ern blot analysis, we were able to show that the IE2-p86 protein can be par tially converted to a 105-kDa isoform in a dose-dependent manner after cotr ansfection of an epitope-tagged SUMO-I, Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the c ovalent coupling of SUMO-1 or hSMT3b to 1E2-p86 both after transient transf ection and after Lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinit y at amino acid positions 175 and 180, which appear to be used alternativel y for coupling, By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. Howe ver, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analys is with the SUMOylation-defective mutant. This suggests a functional releva nce of covalent modification by ubiquitin-homologous proteins for IE2-media ted transactivation, possibly by providing an additional interaction motif for cellular cofactors.