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Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth.

ABSTRACT: The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.

SUBMITTER: Kim ET

PROVIDER: S-EPMC2916544 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth.

Kim Eui Tae ET Kim Young-Eui YE Huh Yong Ho YH Ahn Jin-Hyun JH

Journal of virology 20100602 16

The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growt ...[more]

PMID: 20519406

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Project description:Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

Project description:Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.

Dataset Information

Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth.

Publications

Role of noncovalent SUMO binding by the human cytomegalovirus IE2 transactivator in lytic growth.

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