In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(⌬290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.Human cytomegalovirus (HCMV), a member of the betaherpesvirus subfamily, typically causes nearly ubiquitous asymptomatic latent or persistent infections. However, primary infections of newborns and reactivation from latent infection in immunocompromised individuals, including recipients of organ transplantation and patients with AIDS, can lead to lifethreatening problems with overt systematic and chronic disease (56, 65). During lytic cycle infection, HCMV gene expression occurs in a three-step sequential fashion with immediate-early (IE or ␣), delayed-early (DE or ␤), and late (L or ␥) kinetics. Among the IE proteins, two nuclear regulatory phosphoproteins, IE1 (or IE72) and IE2 (or IE86), are the first and most abundantly expressed proteins and are synthesized by differential splicing from the same complex overlapping transcription unit within the major IE (MIE) locus (56).The 72-kDa IE1 protein (491 amino acids) is encoded by exons 1, 2, 3, and 4 (UL123), whereas the 86-kDa IE2 protein (579 amino acids) is encoded by exons 1, 2, 3, and 5 (UL122), and the two proteins share 85 amino acids at the N terminus. IE2 is a spe...

The human cytomegalovirus (HCMV) 72-kDa immediate-early 1 (IE1) protein is thought to modulate cellular antiviral functions impacting on promyelocytic leukemia (PML) nuclear bodies and signal transducer and activator of transcription (STAT) signaling. IE1 consists of four distinct regions: an amino-terminal region required for nuclear localization, a large central hydrophobic region responsible for PML targeting and transactivation activity, an acidic domain, and a carboxyl-terminal chromatin tethering domain. We found that the acidic domain of IE1 is required for binding to STAT2. A mutant HCMV encoding IE1(⌬421-475) with the acidic domain deleted was generated. In mutant virus-infected cells, IE1(⌬421-475) failed to bind to STAT2. The growth of mutant virus was only slightly delayed at a high multiplicity of infection (MOI) but was severely impaired at a low MOI with low-level accumulation of viral proteins. When cells were pretreated with beta interferon, the mutant virus showed an additional 1,000-fold reduction in viral growth, even at a high MOI, compared to the wild type. The inhibition of STAT2 loading on the target promoter upon infection was markedly reduced with mutant virus. Furthermore, sumoylation of IE1 at this acidic domain was found to abolish the activity of IE1 to bind to STAT2 and repress the interferon-stimulated genes. Our results provide genetic evidence that IE1 binding to STAT2 requires the 55-amino-acid acidic domain and promotes viral growth by interfering with interferon signaling and demonstrate that this viral activity is negatively regulated by a cellular sumoylation pathway.

The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathione S-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection. Human cytomegalovirus (HCMV) can cause severe disease complications and pathogenesis on infection of newborns or immunocompromised individuals, whereas infection of immunocompetent individuals is typically asymptomatic (8, 44).Gene expression during the permissive lytic cycle of HCMV occurs in a three-step sequential fashion. Shortly after infection, the immediate-early (IE) genes are expressed in the absence of de novo protein synthesis. IE proteins and virion factors are required for the subsequent induction of early and late genes (36, 56). The major IE (MIE) locus of HCMV genome encodes two nuclear phosphoproteins, namely, IE1 (UL123, IE72) and IE2 (UL122, IE86), which are translated from differentially spliced mRNA species (58, 60). Some a...