PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.     

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 potent transactivator of viral as well as cellular promoters. Several lines of evidence indicate that this broad transactivation spectrum is mediated by protein-protein interactions. To identify novel cellular binding partners, we performed a yeast two-hybrid screen using a N-terminal deletion mutant of IE2-p86 comprising amino acids 135 to 579 as a bait. Here, we report the isolation 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 hSMT3b have previously been shown to be covalently coupled to a subset of nuclear proteins such as the nuclear domain 10 (ND10) proteins PML and Sp100 in a manner analogous to ubiquitinylation, which we call SUMOylation. By Western blot analysis, we were able to show that the IE2-p86 protein can be partially converted to a 105-kDa isoform in a dose-dependent manner after cotransfection of an epitope-tagged SUMO-1. Immunoprecipitation experiments of the conjugated isoforms using denaturing conditions further confirmed the covalent coupling of SUMO-1 or hSMT3b to IE2-p86 both after transient transfection and after lytic infection of human primary fibroblasts. Moreover, we defined two modification sites within IE2, located in an immediate vicinity at amino acid positions 175 and 180, which appear to be used alternatively for coupling. By using a SUMOylation-defective mutant, we showed that the targeting of IE2-p86 to ND10 occurs independent of this modification. However, a strong reduction of IE2-mediated transactivation of two viral early promoters and a heterologous promoter was observed in cotransfection analysis with the SUMOylation-defective mutant. This suggests a functional relevance of covalent modification by ubiquitin-homologous proteins for IE2-mediated transactivation, possibly by providing an additional interaction motif for cellular cofactors.     

SUMO-1 modification increases human SOD1 stability and aggregation

The mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause approximately 20% cases of familial amyotrophic lateral sclerosis (FALS), characterized by selective loss of motor neurons. Mutant SOD1 forms inclusions in tissues from FALS patients. However, the precise mechanism of the accumulation of mutant SOD1 remains unclear. Here we show that human SOD1 is a substrate modified by SUMO-1. A conversion of lysine 75 to an arginine within a SUMO consensus sequence in SOD1 completely abolishes SOD1 sumoylation. We further show that SUMO-1 modification, on both wild-type and mutant SOD1, increases SOD1 steady state level and aggregation. Moreover, SUMO-1 co-localizes onto the aggregates formed by SOD1. These findings imply that SUMO-1 modification on lysine 75 may participate in regulating SOD1 stability and its aggregation process. Thus, our results suggest that sumoylation of SOD1 may be involved in the pathogenesis of FALS associated with mutant SOD1.     

Covalent modification of human immunodeficiency virus type 1 p6 by SUMO-1

The p6 domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein mediates virion budding from infected cells via protein-protein contacts with the class E vacuolar protein sorting factors, Tsg101 and AIP1/ALIX. Interaction with Tsg101 is strengthened by covalent attachment of monovalent ubiquitin to HIV-1 p6. To identify additional host factors that bind to HIV-1 p6, a human cDNA library was screened in the yeast two-hybrid system. HIV-1 p6 was found to interact with small ubiquitin-like modifier 1 (SUMO-1) as well as the E2 SUMO-1 transfer enzyme, Ubc9. Interaction with p6 was also detected with Daxx, a cellular protein to which SUMO-1 is sometimes covalently attached. SUMO-1 was incorporated into HIV-1 virions where it was protected within the virion membrane from digestion by exogenous protease. Of the two lysine residues in p6, lysine 27 uniquely served as a site of covalent SUMO-1 attachment. As previously reported, though, HIV-1 bearing the p6-K27R mutation replicated just like the wild type. Overproduction of SUMO-1 in HIV-1 producer cells had no apparent effect on virion release or on virion protein or RNA content. Infectivity of the resulting virions, though, was decreased, with the defect occurring after membrane fusion, at the time of viral cDNA synthesis. HIV-1 bearing the p6-K27R mutation was insensitive to SUMO-1 overexpression, suggesting that covalent attachment of SUMO-1 to p6 is detrimental to HIV-1 replication.     

SUMO-1 modification alters ADAR1 editing activity

We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity.     

Human cytomegalovirus IE1 and IE2 proteins block apoptosis.

Human cytomegalovirus-infected fibroblasts are resistant to the induction of apoptosis by superinfection with a mutant adenovirus unable to produce the viral E1B 19-kDa protein that normally causes an E1A protein-mediated apoptotic response. Two cytomegalovirus gene products that block apoptosis were identified. The IE1 and IE2 proteins each inhibit the induction of apoptosis by tumor necrosis factor alpha or by the E1B 19-kDa-protein-deficient adenovirus but not by irradiation with UV light. Our results suggest a new physiological role for the IE1 and IE2 proteins in the human cytomegalovirus replication cycle.     

Interactions between human cytomegalovirus IE1-72 and cellular p107: functional domains and mechanisms of up-regulation of cyclin E/cdk2 kinase activity

Previous work has demonstrated that the human cytomegalovirus IE1-72 protein is able to bind to the N terminus of p107, and IE1-72 alone is sufficient for alleviation of p107-mediated cell growth suppression. However, the mechanism of this alleviation is unclear. Here, we show that IE1-72 can alleviate p107 inhibition of cyclin E/cdk2 kinase activity. We cotransfected various IE1-72 and p107 constructs into C33A cells and demonstrated that IE1-72 could activate the kinase activity of cyclin E/cdk2. Conversely, IE2-86 did not activate this activity, suggesting that the interaction between p107 and IE1-72 and the subsequent kinase activation are specific. By the use of a series of deletion and point mutants of IE1-72 and p107, we observed that a mutation of the loop region of helix-loop-helix-turn-helix in exon 3 of IE1-72 as well as a mutation of the leucine zipper-2 region in exon 4 of IE1-72 abolished binding to p107. In addition, these two IE1-72 mutants did not alleviate p107 inhibition of cyclin E/cdk2 kinase activity and also failed to alleviate p107 inhibition of the E2F-responsive promoter. Meanwhile, deletion of the N-terminal aa 1 to 175 of p107 abolished both p107 binding with IE1-72 and p107 inhibition of cyclin E/cdk2 kinase activity. This result confirms that the N-terminus aa 1 to 175 region of p107 is a common region where both IE1-72 protein and cyclin E/cdk2 bind. We propose a mechanism in which binding of IE1-72 to p107 displaces cyclin E/cdk2 from p107. Once released from p107, cyclin E/cdk2 is able to function as an active kinase.     

Human cytomegalovirus IE1-72 activates ataxia telangiectasia mutated kinase and a p53/p21-mediated growth arrest response

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.     

The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II

In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.