David G. Meckes

Human tumor virus utilizes exosomes for intercellular communication

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.

Microvesicles and Viral Infection

ABSTRACT Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.

Modulation of B-cell exosome proteins by gamma herpesvirus infection

Significance Exosomes are released from tumor cells at high levels, and multiple studies have determined that the secreted exosomes enter recipient cells and can affect their biologic and biochemical properties. In this study, the specific effects of the oncogenic herpesviruses, EBV and Kaposi sarcoma-associated virus, on the proteomes of B-cell exosomes were determined using global quantitative proteomics. The data indicate that the viruses greatly impact the protein content of exosomes with common and distinct changes induced by both viruses. It is likely that these alterations in exosome content modulate the tumor environment, potentially to enhance viral infection and promote tumorigenesis. The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.

Epstein-Barr Virus LMP1 Activates EGFR, STAT3, and ERK through Effects on PKCδ

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the worlds adult population and is linked to multiple malignancies, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). The EBV oncoprotein LMP1 induces transcription of the epidermal growth factor receptor (EGFR), which is expressed at high levels in NPC. EGFR transcription is induced by LMP1 through a p50 NFκB1-Bcl-3 complex, and Bcl-3 is induced by LMP1-mediated activation of STAT3. This study reveals that LMP1, through its carboxyl-terminal activation domain 1 (LMP1-CTAR1), activates both STAT3 and EGFR in a serum-independent manner with constitutive serine phosphorylation of STAT3. Upon treatment with EGF, the LMP1-CTAR1-induced EGFR was additionally phosphorylated and STAT3 became phosphorylated on tyrosine, concomitant with upregulation of a subset of STAT3 target genes. The kinase responsible for LMP1-CTAR1-mediated serine phosphorylation of STAT3 was identified to be PKCδ using specific RNAi, a dominant negative PKCδ, and the PKCδ inhibitor rottlerin. Interestingly, inhibition of PKCδ also inhibited constitutive phosphorylation of EGFR and LMP1-CTAR1-induced phosphorylation of ERK. Inhibition of PKCδ blocked LMP1-CTAR1-mediated transformation of Rat-1 cells, likely through the inhibition of ERK activation. These findings indicate that LMP1 activates multiple distinct signaling pathways and suggest that PKCδ functions as a master regulator of EGFR, STAT3, and ERK activation by LMP1-CTAR1.

ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles.

Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes.

Dynamic interactions of the UL16 tegument protein with the capsid of herpes simplex virus.

ABSTRACT The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines.

Exosomal Communication Goes Viral

ABSTRACT Exosomes are small vesicles secreted from cells that participate in intercellular communication events. Accumulating evidence demonstrates that host exosome pathways are hijacked by viruses and that virally modified exosomes contribute to virus spread and immune evasion. In the case of tumor viruses, recent findings suggest that alterations in normal exosome biology may promote the development and progression of cancer. These studies will be discussed in the context of our current knowledge of Epstein-Barr virus (EBV)-modified exosomes.

Analysis of the Interaction between the UL11 and UL16 Tegument Proteins of Herpes Simplex Virus

ABSTRACT The UL11 and UL16 tegument proteins of herpes simplex virus are conserved throughout the herpesvirus family. Previous studies have shown that these proteins interact, perhaps to link UL16-bound nucleocapsids to UL11, which resides on the cytoplasmic face of the trans-Golgi network, where maturation budding occurs. Little is known about the interaction except that it requires the leucine-isoleucine (LI) and acidic cluster motifs in UL11 and that no other viral proteins are involved. In particular, the important question of whether these two proteins bind to each other directly has not been addressed. Accordingly, UL11 and UL16 were expressed in bacteria, and the purified proteins were found to retain the ability to interact in a manner that was dependent upon the LI and acidic cluster. In an attempt to map the UL11-binding site contained in UL16, a large number of deletion mutants were constructed. The first 40 (nonconserved) amino acids were found to be dispensable, but all the other constructs failed to bind UL11 or had poor expression in transfected cells, suggesting that UL16 is very sensitive to alterations and probably lacks a multidomain structure. As an alternative strategy for identifying residues that are important for the interaction, the cysteines of UL16 were investigated, because many of these are highly conserved. Approximately half of the 20 cysteines in UL16 have been shown to be covalently modified by N-ethylmaleimide, and this treatment was found to block the interaction with UL11. Moreover, individual serine replacements of six of the most conserved cysteine residues were made, and four of these disrupted the interaction with UL11 without affecting protein stability. However, the UL11-UL16 interaction does not involve the formation of interspecies disulfide bonds, because binding occurred even when all the cysteines in UL11 were eliminated. Thus, UL16 directly interacts with UL11 and does so in a manner that requires free cysteines.

Interaction Domains of the UL16 and UL21 Tegument Proteins of Herpes Simplex Virus

ABSTRACT The UL16 protein of herpes simplex virus is capsid associated and was previously identified as a binding partner of the membrane-associated UL11 tegument protein (J. S. Loomis, R. J. Courtney, and J. W. Wills, J. Virol. 77:11417-11424, 2003). In those studies, a less-prominent, ∼65-kDa binding partner of unknown identity was also observed. Mass spectrometry studies have now revealed this species to be UL21, a tegument protein that has been implicated in the transport of capsids in the cytoplasm. The validity of the mass spectrometry results was tested in a variety of coimmunoprecipitation and glutathione S-transferase pull-down experiments. The data revealed that UL21 and UL16 can form a complex in the absence of other viral proteins, even when the assays used proteins purified from Escherichia coli. Moreover, UL11 was able to pull down UL21 only when UL16 was present, suggesting that all three proteins can form a complex. Deletion analyses revealed that the second half of UL21 (residues 268 to 535) is sufficient for the UL16 interaction and packaging into virions; however, attempts to map a subdomain of UL16 were largely unsuccessful, with only the first 40 (of 373) residues being found to be dispensable. Nevertheless, it is clear that UL16 must have two distinct binding sites, because covalent modification of its free cysteines with N-ethylmaleimide blocked binding to UL11 but not UL21. These findings should prove useful for elucidating the molecular machinery used to transmit a signal into a virion when it attaches to cells, a recently discovered mechanism in which UL16 is a central player.

Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus

ABSTRACT The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing UL11 at the UL35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.