Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis

Stinski MF (2013) Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis. PLoS ONE 8(12): e81583. doi:10.1371/journal.pone.0081583 Interaction Network of Proteins Associated with Human Cytomegalovirus IE2-p86 Protein during Infection: A Proteomic Analysis Guixin Du 0 Mark F. Stinski 0 Zhi-Ming Zheng, National Institute of Health - National Cancer Institute, United States of America 0 Department of Microbiology, Carver College of Medicine, University of Iowa , Iowa City, Iowa , United States of America Human cytomegalovirus protein IE2-p86 exerts its functions through interaction with other viral and cellular proteins. To further delineate its protein interaction network, we generated a recombinant virus expressing SG-tagged IE2-p86 and used tandem affinity purification coupled with mass spectrometry. A total of 9 viral proteins and 75 cellular proteins were found to associate with IE2-p86 protein during the first 48 hours of infection. The protein profile at 8, 24, and 48 h post infection revealed that UL84 tightly associated with IE2-p86, and more viral and cellular proteins came into association with IE2-p86 with the progression of virus infection. A computational analysis of the protein-protein interaction network indicated that all of the 9 viral proteins and most of the cellular proteins identified in the study are interconnected to varying degrees. Of the cellular proteins that were confirmed to associate with IE2-p86 by immunoprecipitation, C1QBP was further shown to be upregulated by HCMV infection and colocalized with IE2-p86, UL84 and UL44 in the virus replication compartment of the nucleus. The IE2-p86 interactome network demonstrated the temporal development of stable and abundant protein complexes that associate with IE2-p86 and provided a framework to benefit future studies of various protein complexes during HCMV infection. - Funding: This work was supported by grant AI-13562 from the National Institutes of Health (to M.F.S). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Human cytomegalovirus (HCMV), a prototype b-herpesvirus, causes life-threatening disease in immunocompromised adults, such as AIDS patients and organ transplant recipients, whereas it usually causes asymptomatic persistent infection in healthy individuals. In addition, it is the leading infectious cause of congenital abnormalities and mental retardation in newborns in the United States [1]. Furthermore, chronic HCMV infection has recently been implicated as a cofactor in cardiovascular disease [2] as well as malignant diseases [24]. HCMV only infects humans and replicates preferentially in terminally differentiated cells. Infection progresses through three temporal phases, defined as immediate early (IE), early (E), and late (L). Transcription of the IE genes occurs at five genetic loci and is independent of de novo viral protein synthesis. IE gene products have multiple functions including activating expression of early viral genes, inhibiting apoptosis, and countering intrinsic and innate host immunity [5,6]. Early viral proteins either participate directly in viral DNA synthesis or provide an optimal cellular condition for viral DNA replication. The late genes, which primarily encode structural proteins, are expressed after viral DNA replication [1]. The major immediate-early (MIE) gene locus, a master switch for lytic HCMV infection, generates two predominant viral proteins, IE1-p72 and IE2-p86, and several minor isoforms [6]. While the most abundant MIE protein, IE1-p72, is only required for HCMV replication at low multiplicity of infection (MOI), the less abundant IE2-p86 is essential for viral replication [7,8]. IE2p86 protein has been extensively studied using in vitro methods and multiple functions have been ascribed to it. IE2-p86 binds to a 14base pair cis-repression sequence in the MIE promoter to negatively autoregulate expression of the IE1 and IE2 transcripts [911]. It transactivates viral early gene expression via its interactions with cellular basal transcription machinery [12]. It up-regulates a vast array of cellular genes, including those involved in progression of the cell cycle from G0/G1 to S phase, such as the E2F-responsive genes [13]. However, the IE2-p86 protein also arrests cell cycle progression in both p53-wild type and -null cells [6,14,15]. Moreover, the IE2-p86 protein can block virus-induced chemokine expression [16]. A collection of published reports indicates that IE2-p86 protein can bind to a wide array of different cellular proteins, including pRb, p53, p21, Cdt1, basal transcription factors (TBP, TFIIB, and TAFs), histone acetylases (CBP, p300, and PCAF), histone deacetylases (HDAC1, HDAC2, and HDAC3), histone methyltransferases (G9a and Suvar(3-9)H1), SUMO-1 and Ubc9, mdm2, PIAS1, and Sp1 (reviewed in Stinski and Petrik, 2008) [6]. Although these studies provide insight into the mechanism of IE2p86 function, many of the studies are limited in interaction by extrapolation from results of in vitro binding assays or the forced over-expression of proteins of interest. Nevertheless, IE2-p86 likely exerts many of its biological functions by way of stable as well as transitory protein-protein interactions. There remains a major gap in knowledge as to the temporal sequence of these interactions and which proteins bind to IE2-p86 under normal infected cell conditions. Developments in affinity-purification based isolation methods coupled with mass spectometry (AP-MS) has greatly facilitated identification of proteins in isolated complexes [17]. For example, over 50 cellular proteins were identified to interact with herpes simplex virus early protein ICP8 [18]. The ICP8 interactome is involved in various cellular functions such as viral DNA replication, DNA repair, recombination, and chromatin remodeling. With HCMV, the interacting partners of viral proteins UL84, UL44, UL38, UL29/28, and UL35/35a have been studied using the AP-MS method [1924]. IE2-p86 binds to itself and to the viral protein UL84 to form a complex involved in the initiation of viral DNA synthesis from oriLyt [25]. Gao et al. reported that viral protein UL84 interacts with cellular protein ubiquitinconjugating enzyme E2, casein kinase II, p32 (C1QBP), and importin, as well as viral proteins UL44 and pp65 [24]. Strang et al. detected nucleolin, UL54, IRS1, and UL25 associated with UL44 during the late phase of infection with HCMV [22]. Given the approximately 175 designated open reading frames (ORF) of HCMV and, the approximately 751 putative ORFs identified recently [26], there is much to be learned about the HCMV interactome. In this study, we used tandem affinity purification- mass spectromet (...truncated)