Physical Requirements and Functional Consequences of Complex Formation between the Cytomegalovirus IE1 Protein and Human STAT2

Our previous work has shown that efficient evasion from type I interferon responses by human cytomegalovirus (hCMV) requires expression of the 72-kDa immediate-early 1 (IE1) protein. It has been suggested that IE1 inhibits interferon signaling through intranuclear sequestration of the signal transducer and activator of transcription 2 (STAT2) protein. Here we show that physical association and subnuclear colocalization of IE1 and STAT2 depend on short acidic and serine/proline-rich low-complexity motifs in the carboxy-terminal region of the 491-amino-acid viral polypeptide. These motifs compose an essential core (amino acids 373 to 420) and an adjacent ancillary site (amino acids 421 to 445) for STAT2 interaction that are predicted to form part of a natively unstructured domain. The presence of presumably “disordered” carboxy-terminal domains enriched in low-complexity motifs is evolutionarily highly conserved across all examined mammalian IE1 orthologs, and the murine cytomegalovirus IE1 protein appears to interact with STAT2 just like the human counterpart. A recombinant hCMV specifically mutated in the IE1 core STAT2 binding site displays hypersensitivity to alpha interferon, delayed early viral protein accumulation, and attenuated growth in fibroblasts. However, replication of this mutant virus is specifically restored by knockdown of STAT2 expression. Interestingly, complex formation with STAT2 proved to be entirely separable from disruption of nuclear domain 10 (ND10), another key activity of IE1. Finally, our results demonstrate that IE1 counteracts the antiviral interferon response and promotes viral replication by at least two distinct mechanisms, one depending on sequestration of STAT2 and the other one likely involving ND10 interaction.Human cytomegalovirus (hCMV) is an extremely widespread opportunistic pathogen causing morbidity and mortality in hundreds of thousands of children and adults each year (48). Within the ∼230,000-bp hCMV genome, the major immediate-early (IE) gene is believed to have a decisive role in acute infection and reactivation from viral latency. Through differential splicing, polyadenylation, and promoter usage, this viral genomic region produces multiple mRNAs. Although a variety of protein products expressed from these mRNAs have been identified (4, 55), the UL123-coded 72-kDa nuclear phosphoprotein IE1 and the UL122-coded 86-kDa nuclear phosphoprotein IE2 are the most abundant and important. They share 85 amino-terminal amino acids corresponding to major IE exons 2 and 3 but have distinct carboxy-terminal parts, encoded by exon 4 (IE1) or exon 5 (IE2). Both proteins have long been recognized as promiscuous transcriptional regulators. IE2 is the principal activator of the hCMV lytic cycle and is essential for productive viral replication (26, 40). The role of IE1 in hCMV infection is less clear than that of IE2. Whereas IE1-null viruses replicate efficiently in fibroblasts at high input multiplicities, the absence of IE1 results in inefficient hCMV early gene expression and attenuated viral growth under “single-hit” conditions (20, 22, 47). Consistent with its role in transcriptional activation, IE1 has been reported to interact with several transcriptional coactivators (25, 30, 41, 56, 84) and a histone deacetylase (28, 51). A small fraction of IE1 is also found covalently conjugated to the small ubiquitin-like modifier 1 (SUMO-1) (28, 36, 49, 50, 67). Furthermore, the viral protein has the remarkable ability to localize to both the chromatin (33) and the interchromatinic matrix-associated nuclear domain 10 (ND10) compartments of the cell nucleus. IE1 binds to at least one constituent of ND10, namely, the promyelocytic leukemia (PML) protein, and disrupts these structures upon ectopic expression or at early times after hCMV infection (2, 32, 78). It has recently been demonstrated that individual ND10 components, including PML, Sp100, and Daxx, mediate an intrinsic immune response against hCMV and other herpesviruses (18, 19, 60, 61, 71, 72, 79). This observation supports the idea that ND10-resident proteins are part of a cellular antiviral defense mechanism which is inactivated by virus-encoded proteins, including IE1 (3, 71, 72; reviewed in references 17, 42, and 73). The IE1 nucleotide and protein sequences are evolutionarily conserved among primate CMVs. In contrast, rodent CMVs have positional IE1 orthologs that share no obvious amino acid sequence similarity with the hCMV counterpart. Nonetheless, the hCMV and murine CMV (mCMV) IE1 proteins exhibit discrete as well as common functional activities (reviewed in references 7 and 43).In addition to its proposed role in antagonizing ND10-related cellular defense mechanisms, hCMV IE1 also inactivates a crucial branch of the host''s inducible innate immune system. We have demonstrated that the viral protein inhibits type I interferon (IFN) signaling, conferring a substantial degree of protection against the antiviral effects of IFN-α and IFN-β upon hCMV (53). Remarkably, IE1 interferes with one of the final steps in the Janus kinase-signal transducer and activator of transcription (Jak-STAT) cascade, which links the cytoplasmic membrane-bound type I IFN receptor to the promoters of IFN-stimulated genes (ISGs), many of which encode antiviral proteins or RNAs (reviewed in reference 62). In particular, the viral protein prevents sequence-specific promoter binding and transcriptional activation by the IFN-stimulated gene factor (ISGF3) complex, which forms in the presence of IFN-α or -β (53). ISGF3 is composed of three proteins: STAT1, STAT2, and IFN regulatory factor 9 (IRF9, also known as p48 or ISGF3γ), and IE1 was found to be physically associated with two of these components (STAT1 and STAT2) (28, 53). However, STAT2 appears to be the viral protein''s primary target in the trimeric complex (S. Krauss, S. Meinel, I. Tschertner, C. Paulus, and M. Nevels, unpublished data). Despite the fact that STAT2 binding is expected to contribute substantially to the overall function of IE1 during hCMV infection, we have only just begun to understand the structural basis for and relevance of this interaction (28).Here we define the structural requirements in the hCMV IE1 protein that contribute to physical and functional interaction with STAT2. We present evidence that STAT2 binding is evolutionarily conserved between mammalian CMV IE1 proteins. Moreover, we show for the first time that STAT2 binding and ND10 disruption are genetically separable activities of IE1 and that STAT2 interaction is one of at least two distinct mechanisms by which the viral protein antagonizes the antiviral IFN response to promote viral replication.