LMP1 association with CD63 in endosomes and secretion via exosomes limits constitutive NF-κB activation.

The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in ∼90% of the world population. Constitutive activation of NF-κB by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-κB overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-κB activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) cells.

Possible contributing role of Epstein-Barr virus (EBV) as a cofactor in human papillomavirus (HPV)-associated cervical carcinogenesis.

BACKGROUND: Persistent infection with EBV has been linked to the development of malignancies including HPV-associated cervical carcinoma. However, the role of EBV in HPV-associated cervical cancer is still poorly understood. OBJECTIVE: To determine the possible contributing role of EBV in HPV-associated cervical carcinogenesis according to HPV genotypes, HPV genome status and EBV localization. STUDY DESIGN: Cervical tissues, including 82 with no squamous intraepithelial lesions (noSILs), 85 low-grade SILs (LSILs), 85 high grade SILs (HSILs) and 40 squamous cell carcinoma samples (SCC) were investigated using PCR and dot blot hybridization for EBV detection and PCR and reverse line blot hybridization for HPV genotyping. The amplification of papillomavirus oncogene transcripts assay and in situ hybridization were used to determine HPV physical status and EBV EBER localization, respectively. RESULTS: EBV was detected increasingly from noSIL (13.4%), LSIL (29.4%) to HSIL (49.4%) samples. The prevalence of HPV-EBV co-infection was significantly higher in any grade of lesion than in noSIL samples (p<0.05) including noSIL (1.2%; 95% confidence intervals [CI]=0.0-3.6%, relative risk [RR]=1), LSIL (18.8%, 95% CI=10.5-27.1%, RR=15.4), HSIL (41.2%, 95% CI=30.7-51.6%, RR=33.8) and SCC (30.0%, 95% CI=15.8-44.2%, RR=24.6). Interestingly, HPV-EBV co-infection was more common in cases with episomal forms of high-risk (HR) HPV whereas HPV alone was more common in cases with integrated HR-HPV. In addition, EBER staining demonstrated that EBV was mainly present in infiltrating lymphocytes. CONCLUSION: Infiltrating EBV-infected lymphocytes may play a role in cancer progression of cervical lesion containing episomal HR-HPV.

Exosomes: Fit to deliver small RNA.

Exosomes are specialized membranous nano-sized vesicles derived from endocytic compartments that are released by many cell types. Microvesicles are distinctive from exosomes in that they are produced by shedding of the plasmamembrane and usually larger in size (>1 µm). Exosome biogenesis involves the tightly controlled process of inward budding from the limiting membrane of multivesicular bodies (MVBs). This results in numerous intraluminal vesicles in the lumen of MVBs that contain distinct protein repertoires. It has been suggested that microvesicles shed by certain tumor cells hold functional messenger RNA (mRNA) that may promote tumor progression. We discovered that purified exosomes contain functional microRNAs (miRNAs) and small RNA, but detected little mRNA. Although a clear and decisive distinction between microvesicles and exosomes cannot be made and different subsets of exosomes exist, we speculate that exosomes are specialized in carrying small RNA including the class 22-25 nucleotide regulatory miRNAs. To demonstrate this we developed a co-culture system and found that exosomes are continuously secreted and transferred from Epstein Barr virus (EBV)-infected cells to uninfected neighboring cells. Throughout exosome transfer, the exogenous EBV-encoded miRNAs were delivered to subcellular sites of miRNA-mediated gene repression. Additionally, we found evidence that mature miRNAs are transferred between circulating cells in humans, since we detected EBV-miRNAs in non-infected cells in the peripheral blood of patients that include monocytes and T cells. In this addendum we discuss these findings in the context of recently published papers that advanced our current knowledge of exosome physiology, (mi)RNA function and intercellular RNA transfer. Based on this information we propose that an intercellular (miRNA-based) mode of signal transmission may be well suited in controlling space-confined processes such as the initiation of immune responses in the secondary (peripheral) lymphoid tissues or in a tumor microenvironment. Deciphering the molecular mechanism(s) that control small RNA loading into exosomes and transfer to recipient cells in vitro will provide new evidence for the physiological relevance of vesicle-mediated intercellular communication in vivo.