Modulation of B-cell exosome proteins by gamma herpesvirus infection

Abstract

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.

Keywords: microparticles; microvesicles; oncosomes.

Fig. 1.

Characterization of exosomes secreted from B cells. (A) Whole-cell lysates from various B-cell lines that were uninfected (BJAB), EBV infected (#1, HLJ, IM9, CP), KSHV infected (JC, BCBL1, BCP1, BC3), or dually infected (BC1 and JSC-1) were separated by SDS/PAGE and analyzed by immunoblot analysis for the indicated proteins. GAPDH was used as a loading control. (B) Exosomes were purified from the conditioned media of the B cells and analyzed by electron microscopy. Representative images of exosomes produced from the IM9 and BC1 cell lines are shown. (C) Lysates from purified exosomes were separated by SDS/PAGE and analyzed by immunoblot for expression of indicated viral proteins and common exosome markers. Asterisks indicate a with a higher molecular weight detected using EBNA2 antibody.

Fig. 2.

Venn diagrams of proteins identified in B-cell exosomes by mass spectrometry. (A) The total number of proteins identified throughout all B-cell exosome samples was compared with results from the entire Exocarta database of published exosome proteomics. (B) Exosome components identified from the EBV- or KSHV-infected cells were compared with those found in the uninfected BJAB control cell line. (C) Comparison of proteins specific to each infection type and those proteins common to infection types.

Fig. 3.

2D DIGE and Decyder analysis of B-cell exosome proteomes. Exosomal proteins were labeled with fluorescent dyes and separated by 2D DIGE in pH 3–10 immobilized gradients and SDS 12.5% polyacrylamide gels. (A) Representative gel shown (1 of 10) with IM9 exosome proteins labeled with Cy3 (green) and BC-3 exosome proteins labeled with Cy5 (red). PCA was performed on the 2D DIGE data and plotted based on (B) LMP1 expression or (C) viral groups. (D) Hierarchical cluster analysis of significantly (P ≤ 0.05, ANOVA) altered exosome components grouped by viral infection status. Red lines represent proteins whose expression levels were up-regulated relative to the group, green lines represent down-regulated proteins, and black lines represent unchanged proteins. The red triangle depicts relative LMP1 expression in the EBV cell lines.

Fig. 4.

Hierarchical clustering of B-cell exosome proteins. (A) Unsupervised hierarchical clustering of B-cell exosome proteins that show up-regulation in EBV-infected B-cell lines as determined by spectral count-based quantitative proteomic analysis. (B) Unsupervised hierarchical clustering of B-cell exosome proteins that show up-regulation in KSHV- or KSHV+EBV-infected cell lines that also were LMP1⁻.

Fig. 5.

Label-free spectral count-based quantitative proteomic analysis. (A) Volcano plot showing normalized differences in protein expression in LMP1⁺ and LMP1⁻ B-cell lines infected with EBV, KSHV, or EBV+KSHV. Note that the ANOVA P value of 0.05 (−log₁₀ = 1.3) is used to identify significant differences in protein expression. (B) Enlarged view of the region highlighted in orange in A. (C) Overall differences in protein expression in LMP1⁺ and LMP1⁻ B-cell lines ranked by protein ID. (Insets) Number of down-regulated and up-regulated proteins that show a log₂ twofold change in the LMP1⁺/LMP1⁻ ratio. Points marked in red represent proteins highlighted in A and B. Infinity units were given arbitrary values of log₂ 5 and −2.5.

Fig. 6.

IPA of virally modified exosome components. IPA of (A) proteins only identified within EBV and KSHV exosomes compared to the BJAB control, (B) genes significantly altered by KSHV and KSHV/EBV relative to BJAB, (C) genes significantly altered by KSHV relative to BJAB, and (D) genes significantly changed when grouped by LMP1 expression. The graphs for the top Diseases and Disorders (Left) and Molecular and Cellular Functions (Center) categories show the total number of molecules identified by mass spectrometry that fall into the designated Ingenuity classification. The Top Canonical Pathways graphs (Right) represent the ratio of the number of molecules identified in this study to the total number of proteins in each Ingenuity pathway.