Identification Of Cellular Proteins Research Articles

Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

New method of extraction of intact nuclei from cells for flow cytometry fluorescence immunoassay

Background. Identification of cellular proteins can be performed by indirect immunofluorescence assay using flow cytometry. However, this method allows to detect cellular proteins that are either on the cell surface or inside the cell and cannot demonstrate a protein distribution within cellular compartments, particularly in nucleus. Meanwhile, the nuclear localization of the protein of interest in many respects gives an indication of the mechanisms of its action. Therefore, the development of the protocol of extraction of nuclei suitable for analysis on a flow cytometer is able to complement the information about the localization and functional significance of many nucleus-associated proteins. Objective. The aim of this study was to develope a method for extraction, purification and stabilization of intact nuclei suitable for flow cytofluorimetry analysis. Materials and methods. In this work we studied the nuclear localization of the receptor type 1 for vascular endothelial growth factor (VEGF-R1). The A431 human cancer cell line was used as an object of the study. The quality of extracted nuclei was assessed by microscopic examination of stained smears of nuclear suspension. Expression of the receptor was determined by indirect immunofluorescence assay using flow cytometry. Results. New method was successfully applied to obtain the suspension of intact cellular nuclei that is crucial to perform further flow cytometry. Applied method revealed the presence of the receptor type 1 for vascular endothelial growth factor at the external nuclear membrane and inside of the nucleus. Interesting to note that the presence of the receptor type 1 for vascular endothelial growth factor inside ofthe nucleus was 3,8 times as much as its surface location (17,9 ± 1,04 % and 4,8 + 0,26 % respectively). Conclusions. The new method of extraction, purification and stabilization ofthe nuclei is applicable for proteins identification by flow cytometry. In combination with other methods (ICC, Western blotting, etc.) flow cytometry of intact nuclei is able to complement the information about the properties of nucleus-associated proteins.

Non-Metabolic Role of PKM2 in Regulation of the HIV-1 LTR.

Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPβ, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning -120 bp to -80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517-525, 2017. © 2016 Wiley Periodicals, Inc.

Nucleoprotein of influenza A virus negatively impacts antiapoptotic protein API5 to enhance E2F1-dependent apoptosis and virus replication.

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

Dengue virus NS1 protein interacts with the ribosomal protein RPL18: This interaction is required for viral translation and replication in Huh-7 cells

Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA)

Although it is well documented that the initial attachment receptors for Newcastle Disease Virus (NDV) and Respiratory Syncytial Virus (RSV) are sialic acid-containing molecules and glycosaminoglycans respectively, the exact nature of the receptors for both viruses remains to be deciphered. Moreover, additional molecules at the host cell surface might be involved in the entry mechanism. With the aim of identifying the cellular proteins that interact with NDV and RSV at the cell surface, we performed a virus overlay protein binding assay (VOPBA). Cell membrane lysates were separated by two dimensional (2D) gel electrophoresis and electrotransferred to PVDF membranes, after which they were probed with high viral concentrations. NDV interacted with a Protein Disulfide Isomerase from chicken fibroblasts. In the case of RSV, we detected 15 reactive spots, which were identified as six different proteins, of which nucleolin was outstanding. We discuss the possible role of PDI and nucleolin in NDV and RSV entry, respectively.

Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future.

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ZBRK1 represses HIV-1 LTR-mediated transcription

The identification of cellular proteins that interact with the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) provides a basic understanding of HIV-1 gene expression, which is the major determinant regulating virus replication. We show that ZBRK1 negatively regulates the HIV-1 LTR. Ectopic expression of ZBRK1 represses transcriptional activity of the HIV-1 LTR, whereas the depletion of endogenous ZBRK1 leads to activation of the HIV-1 LTR. The repressor activity of ZBRK1 is required for TRIM28 binding. Furthermore, ZBRK1 is bound to the HIV-1 LTR in vivo. These results indicate that ZBRK1 could be involved in a potent intrinsic antiretroviral defense. Structured summary of protein interactionsZBRK1physically interacts with TRIM28 by anti tag coimmunoprecipitation (View interaction).

Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.

Identification of Cellular Proteins That Maintain Retroviral Epigenetic Silencing: Evidence for an Antiviral Response

Integrated retroviral DNA is subject to epigenetic gene silencing, resulting in loss of expression of viral genes as well as reporter or therapeutic genes transduced by retroviral vectors. Possible mediators of such silencing include the histone deacetylase (HDAC) family of cellular proteins. We previously isolated HeLa cell populations that harbored silent avian sarcoma virus-based green fluorescent protein (GFP) vectors that could be reactivated by treatment with HDAC inhibitors. Here, we developed a small interfering RNA (siRNA)-based approach to identify specific host factors that participate in the maintenance of silencing. Knockdown of HDAC1, the transcriptional repressor Daxx (a binding partner of HDAC1), or heterochromatin protein 1 gamma resulted in robust and specific GFP reporter gene reactivation. Analyses of cell clones and diverse GFP vector constructs revealed that the roles of HDAC1 and Daxx in retroviral silencing are largely independent of the integration site or the promoter controlling the silent GFP reporter gene. Previous findings from our laboratory and those of others have suggested that Daxx and HDAC proteins may act broadly as part of an antiviral response to repress viral gene transcription. Expression of presumptive viral "countermeasure" proteins that are known to inhibit Daxx or HDACs (pp71, IE2, and Gam1) resulted in the reactivation of GFP reporter gene expression. This study has identified individual host factors that maintain retroviral silencing and supports the proposal that these factors participate in an antiviral response. Furthermore, our results indicate that siRNAs can be used as specific reagents to interrupt the maintenance of epigenetic silencing.

Functional Proteomic Screen Identifies a Modulating Role for CD44 in Death Receptor–Mediated Apoptosis

Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.

Identification of cellular proteins targeted by reactive sulphonamide metabolites and their relation to adverse drug reactions

Clinical Pharmacology & Therapeutics (2003) 73, P10–P10; doi:

Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line.

The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels.

Combination DNA/RNA Fish and Immunophenotyping

This unit presents methods for combining immunophenotyping with DNA/RNA FISH. The approach is used in so-called genotype/phenotype analysis to identify chromosomal aberrations in sub-populations of cells present in heterogenous populations. Combining RNA and DNA detection with identification of cellular proteins is quite difficult. This series of protocols is provided to enable the successful application of the combination of these techniques.

Identification of cellular proteins, that bind selectivity to the conserved 3′ terminus of the hepatitis C virus genome

Identification of cellular proteins, that bind selectivity to the conserved 3′ terminus of the hepatitis C virus genome

Molecular Determinants of Nef Function.

The human immunodeficiency virus (HIV), the causative agent of the acquired immune deficiency syndrome (AIDS), in addition to encoding for the gag, pol and env structural genes common to all retroviruses also encodes six accessory genes: tat, rev, nef, vpr, vpu and vif. These accessory genes are responsible for the regulation of HIV replication. Recent advances in our understanding of the function(s) of these genes have illustrated the complex interplay between HIV, the infected cell and the host. In addition, identification of cellular proteins interacting with accessory gene products have provided new tools to study cellular processes. The topic of this review, nef, has been shown in vitro to induce the cell surface downregulation of CD4, the receptor for HIV, to enhance the infectivity of HIV particles and to associate with at least one cellular serine/threonine kinase. In vivo, Nef is essential for the efficient virus replication responsible for disease progression. In this review, several prominent aspects of Nef function are discussed including its effect on CD4 trafficking, on signaling pathways and on virus infectivity enhancement. Copyright 1997 S. Karger AG, Basel

Identification of Cellular Protein That Can Interact Specifically with the Basic Helix-Loop-Helix Domain of the Aromatic Hydrocarbon Receptor

The Ah receptor is a basic helix-loop-helix (bHLH)-PAS protein and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to xenobiotic responsive elements. We have sought to identify possible cellular proteins that can interact with the Ah receptor. The bHLH domain of the Ah receptor was fused to glutathione-S-transferase (GST), and the resulting fusion protein was used as a probe to help us to identify receptor associated protein(s). At least one such protein, 45kDa (p45), was detected in mouse liver extracts, but it does not bind to the bHLH domain of the Ah receptor nuclear translocator, nor to the transactivation domain of Ah receptor or GST alone.

Identification of Cellular Proteins That Bind the Central Conserved Region of p53

The bacterial fusion protein between glutathione S-transferase and the central conserved region of human p53(GST-p53) was purified and fixed on the beads and then used in the binding assay with radiolabeled cell extract from human hepatocarcinoma cell line, Hep3B. The binding asssay disclosed the presence of cellular proteins that interact with GST-p53 but not with GST. SV40 large T antigen abrogated the bindings of two cellular proteins with molecular weights of 50 kda and 40 kda. The binding of the proteins to p53 was observed in a cell cycle-dependent manner. These two proteins are candidate cellular proteins which regulate the function of p53.

Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro: a role for myristylation

The human immunodeficiency virus (HIV) type 1 nef gene product was expressed as an N-terminal fusion protein with glutathione-S-transferase (GST) in the baculovirus system. The resulting nefGST fusion protein was found to be authentically myristylated at the N terminus and could be purified to homogeneity by one-step affinity chromatography on immobilized glutathione. The high affinity of nefGST for glutathione was exploited to develop an assay to identify cellular proteins capable of interacting with nef. Several such proteins were identified in extracts from the Jurkat human T cell line. The interaction between nef-binding proteins and immobilized nefGST could be specifically competed by the addition of soluble nef. Cell fractionation showed that nef-binding proteins were present in both cytosolic and membrane-associated fractions. A non-myristylated derivative failed to bind to the membrane-associated proteins but was able to bind to the cytosolic group, albeit with reduced affinity. In addition, a single protein present in both soluble and membrane-associated fractions exhibited myristylation-independent binding to nef. By analogy with other myristylated proteins such as MARCKS (myristylated alanine-rich C kinase substrate) and the Rous sarcoma virus transforming protein, src, the membrane-associated proteins that bind only to myristylated nef may represent a specific membrane target for nef. The cytosolic proteins that interact with nef may constitute soluble components of an as yet unidentified signal transduction pathway which is the target of nef action in the HIV-1-infected cell.