Purification Des Proteines Fusinnées Avec La GST

Human tumor virus utilizes exosomes for

intercellular communication

David G. Meckes, Jr.a, Kathy H. Y. Shaira, Aron R. Marquitza, Che-Pei Kungb, Rachel H. Edwardsa,

and Nancy Raab-Trauba,b,1

aThe Lineberger Comprehensive Cancer Center, and bDepartment of Microbiology–Immunology, University of North Carolina, Chapel Hill, NC 27599

Edited* by Elliott Kieff, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, and approved October 15, 2010 (received for review

September 21, 2010)

The Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) is

expressed in multiple human malignancies and has potent effects

on cell growth. It has been detected in exosomes and shown to

inhibit immune function. Exosomes are small secreted cellular

vesicles that contain proteins, mRNAs, and microRNAs (miRNAs).

When produced by malignant cells, they can promote angiogenesis,

cell proliferation, tumor-cell invasion, and immune evasion. In this

study, exosomes released from nasopharyngeal carcinoma (NPC)

cells harboring latent EBV were shown to contain LMP1, signal

transduction molecules, and virus-encoded miRNAs. Exposure to

these NPC exosomes activated the ERK and AKT signaling pathways

in the recipient cells. Interestingly, NPC exosomes also contained

viral miRNAs, several of which were enriched in comparison with

their intracellular levels. LMP1 induces expression of the EGF receptor

in an EBV-negative epithelial cell line, and exosomes produced

by these cells also contain high levels of EGF receptor in exosomes.

These findings suggest that the effects of EBV and LMP1 on cellular

expression also modulate exosome content and properties. The

exosomes may manipulate the tumor microenvironment to influence

the growth of neighboring cells through the intercellular

transfer of LMP1, signaling molecules, and viral miRNAs.

oncogene | herpesvirus

The Epstein–Barr virus (EBV) is a major human pathogen that

potently affects cell growth regulation and is linked to the development

of multiple malignancies (1). These cancers contain the

viral genome but have different patterns of viral gene expression.

Latent membrane protein 1 (LMP1) is considered the major oncogene

of EBV because it has transforming properties in cultured

cell lines, is essential for B-lymphocyte transformation, and is frequently

expressed in EBV-associated cancers (2, 3). LMP1 functions

as a constitutively active member of the tumor necrosis

factor receptor family and activates multiple signaling pathways,

including mitogen-activated protein kinase (MAPK), c-Jun Nterminal

kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt,

and NF-κB (4). LMP1 induces the expression of specific genes that

are involved with apoptosis, cell-cycle progression, cell proliferation,

and migration (4). One important target of LMP1 is epidermal

growth factor receptor (EGFR), a member of the ErbB

receptor tyrosine kinase family (5). Similarly to LMP1, EGFR

activates multiple signaling pathways, including Src kinases, JAKs/

STATs, Ras/MAPK, and PI3K/Akt (6).As a potent growth-signaling

receptor, theEGFRpathway is targeted by several oncogenic viruses,

including EBV, to affect cell growth (6). EBV-positive nasopharyngeal

carcinoma (NPC) has elevated amounts of EGFR that are directly

related to the expression level of LMP1 (5). EGFR can be

secreted from cells in exosomes and other microvesicles, and its

subsequent uptake by endothelial cells can induce tubule formation,

activation of MAPK and Akt pathways, and VEGF expression (7).

Interestingly, glioblastoma microvesicles contain a truncated oncogenic

form of EGFR that induces proliferation of a human glioma

cell line, suggesting potential autocrine and paracrine stimulation (8).

Exosomes are 40- to 100-nm endosomal-derived vesicles that

are secreted from many cell types and transfer proteins, mRNAs,

and microRNAs (miRNAs) to neighboring or distant cells to

modulate immune function, angiogenesis, cell proliferation, tumorcell

invasion, and cell-to-cell communication (9, 10). Exosomes are

present in many biological fluids, including the cerebrospinal fluid,

blood, and urine, and they likely affect physiologic processes (9, 10).

Exosomes are a recently discovered mechanism through which

cancer cells and virally infected cells can manipulate their microenvironment.

Viruses can use the exosome pathway for virus egress

and immune evasion (11–13). Interestingly, EBV-infected cells

release

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