Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication.

Section: Discussionmentioning

confidence: 99%

Foot-and-Mouth Disease Virus Modulates Cellular Vimentin for Virus Survival

Gladue

O’Donnell

Baker-Branstetter

et al. 2013

“…Previous studies have demonstrated both an interaction between LMP1 and the intermediate filament protein vimentin and the regulation of vimentin by LMP1 (34,35). In addition, although previous studies have indicated that LMP1 signaling induces actin cytoskeleton rearrangement, motility, and invasion (36), the direct regulation of LMP1 signaling by the actin cytoskeleton has not been ob- served.…”

Section: Resultsmentioning

confidence: 86%

Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins

Holthusen

Talaty

Everly

2015

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton. IMPORTANCELMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins. E pstein-Barr virus (EBV) is a DNA tumor virus and an etiologic agent of infectious mononucleosis (1, 2). Current models suggest that lytic replication occurs in epithelial cells and that latent infection occurs primarily in B lymphocytes. Latent infection of B lymphocytes with EBV induces a number of cellular changes that reprogram the latently infected cells to establish a subset of memory B cells that contain the viral genome and persist for the life of the infected host. In vitro infection of peripheral blood mononuclear cells with EBV is sufficient to establish latently infected, immortalized lymphocyte cell lines (LCLs). In addition, latent infection is associated with human cancer (3-9). Nearly all patients with endemic cases of Burkitt's lymphoma and nasopharyngeal carcinoma contain EBV, and a significant number of patients with Hodgkin's lymphoma and gastric carcinoma contain EBV. Finally, in the presence of immunodeficiency, EBV induces lymphoproliferative diseases.During lytic replication, EBV expresses the cadre of herpesvirus genes required to replicate the viral DNA and assemble virus particles de novo. During latency, a number of EBV nuclear antigens (EBNAs), latent membrane proteins (LMPs), and regulatory RNAs are expressed (1, 2). These regulate the latently infected cells to alter the cellular environment to favor the survival of the infected cells and the maintenance of the viral episome. LMP1 is required for the establishment of latency in vitro and is considered the oncogene of EBV since it can induce the phenotypic transformation of rodent fibroblasts (10-15). Fibroblasts that exp...

“…Other antibodies used for immunoblotting included anti-LMP1 CS1-4 (Dako, CA, USA); anti-HA (Covance, NC, USA); anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase), anti-IB␣, anti-Myc, and anti-TRAF6 (Santa Cruz Biotechnology); and anti-phospho-Akt and anti-Akt (Cell Signaling Technology). Immunoblotting was conducted as described previously (33). Briefly, protein samples (either immunoprecipitations or 25 g of total cell lysates as determined by the Bradford assay) in Laemmli loading buffer were loaded onto a 12% agarose SDS-polyacrylamide gel.…”

Section: Methodsmentioning

confidence: 99%

“…LMP1 expression was not detected in parental 293T, Rat1, NP69, or MCF10a cells (data not shown). In contrast, the C666 cell line contains the EBV genome and is thought to express LMP1 at low levels (33,36). Parental C666 cells expressed approximately 20% as much LMP1 as C15 tumors (Fig.…”

Section: Lmp1 Expression Increases Igf1r Activationmentioning

confidence: 99%

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LMP1 Promotes Expression of Insulin-Like Growth Factor 1 (IGF1) To Selectively Activate IGF1 Receptor and Drive Cell Proliferation

Tworkoski

Raab‐Traub

2015

Self Cite

Epstein-Barr Virus (EBV) is a gammaherpesvirus that infects the majority of the human population and is linked to the development of multiple cancers, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the primary oncoprotein of EBV, and in epithelial cells it induces the expression and activation, or phosphorylation, of the epidermal growth factor receptor kinase. To identify effects on additional kinases, an unbiased screen of receptor tyrosine kinases potentially activated by LMP1 was performed. Using a protein array, it was determined that LMP1 selectively activates insulin-like growth factor 1 receptor (IGF1R). This activation takes place in fibroblast, epithelial, and nasopharyngeal cell lines that express LMP1 stably and transiently. Of note, LMP1 altered the phosphorylation, but not the expression, of IGF1R. The use of LMP1 mutants with defective signaling domains revealed that the C-terminal activating region 2 domain of LMP1 increased the mRNA expression and the secretion of the ligand IGF1, which promoted phosphorylation of IGF1R. IGF1R phosphorylation was dependent upon activation of canonical NF-B signaling and was suppressed by IB␣ and a dominant negative form of TRAF6. Inhibition of IGF1R activation with two small-molecule inhibitors, AG1024 and picropodophyllin (PPP), or with short hairpin RNA (shRNA) directed against IGF1R selectively reduced proliferation, focus formation, and Akt activation in LMP1-positive cells but did not impair LMP1-induced cell migration. Expression of constitutively active Akt rescued cell proliferation in the presence of IGF1R inhibitors. These findings suggest that LMP1-mediated activation of IGF1R contributes to the ability of LMP1 to transform epithelial cells. IMPORTANCEEBV is linked to the development of multiple cancers in both lymphoid and epithelial cells, including nasopharyngeal carcinoma. Nasopharyngeal carcinoma is a major cancer that develops in specific populations, with nearly 80,000 new cases reported annually. LMP1 is consistently expressed in early lesions and continues to be detected within 50 to 80% of these cancers at later stages. It is therefore of paramount importance to understand the mechanisms through which LMP1 alters cell growth and contributes to tumorigenesis. This study is the first to determine that LMP1 activates the IGF1R tyrosine kinase by regulating expression of the ligand IGF1. Additionally, the data in this paper reveal that specific targeting of IGF1R selectively impacts LMP1-positive cells. These findings suggest that therapies directed against IGF1R may specifically impair the growth of EBV-infected cells. E pstein-Barr Virus (EBV) is a gammaherpesvirus transmitted through bodily fluids that infects both lymphocytes and oropharyngeal epithelial cells. It is estimated that greater than 90% of the human population are EBV carriers, and EBV infection is an etiological factor in the development of multiple cancers such as Burkitt lymphoma, Hodgkin lymphoma, gastric carcinoma, and nasopharynge...