Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection. Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, questions remain regarding the mechanism of EBOV entry. Here, we demonstrate that internalization of EBOV particles is independent of clathrin- or caveolae-mediated endocytosis. Specifically, we show that internalized EBOV particles co-localize with macropinocytosis-specific endosomes (macropinosomes) and that their entry is negatively affected by treatment with macropinocytosis inhibitors. Moreover, the internalization of Ebola virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. We further demonstrate that a pseudotyped vesicular stomatitis virus possessing the EBOV glycoprotein (GP) also co-localizes with macropinosomes and its internalization is similarly affected by macropinocytosis inhibitors. Our results indicate that EBOV uptake into cells involves the macropinocytic pathway and is GP-dependent. These findings provide new insights into the lifecycle of EBOV and may aid in the development of therapeutics for EBOV infection.

Epstein-Barr virus (EBV), a human gammaherpesvirus, establishes a lifelong latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence suggest that exosomes derived from EBV-infected cells are internalized and transfer viral factors, including EBV-encoded latent membrane protein and microRNAs, to the recipient cells. However, the detailed mechanism by which exosomes are internalized and their physiological impact on the recipient cells are still poorly understood. In this study, we visualized the internalization of fluorescently labeled exosomes derived from EBV-uninfected and EBV-infected B cells of type I and type III latency into EBV-negative epithelial cells. In this way, we demonstrated that exosomes derived from all three cell types were internalized into the target cells in a similar fashion. Internalization of exosomes was significantly suppressed by treatment with an inhibitor of dynamin and also by the knockdown of caveolin-1. Labeled exosomes were colocalized with caveolae and subsequently trafficked through endocytic pathways. Moreover, we observed that exosomes derived from type III latency cells upregulated proliferation and expression of intercellular adhesion molecule 1 (ICAM-1) in the recipient cells more significantly than did those derived from EBV-negative and type I latency cells. We also identified the EBV latent membrane protein 1 (LMP1) gene as responsible for induction of ICAM-1 expression. Taken together, our data indicate that exosomes released from EBV-infected B cells are internalized via caveola-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells through transferring one or more viral factors.

We investigated whether Epstein–Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)‐α. EBV‐negative subclones isolated from EBV‐positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN‐α treatment. On the other hand, EBV‐positive counterparts exhibited striking resistance against IFN‐α‐induced apoptosis. Transfection of an individual EBV latent gene into EBV‐negative BL cells revealed that EBV‐encoded poly(A)− RNAs (EBERs) were responsible for IFN resistance. EBERs bound double‐stranded (ds) RNA‐activated protein kinase (PKR), a key mediator of the antiviral effect of IFN‐α, and inhibited its phosphorylation. Transfection of dominant‐negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV‐negative BL cells resistant to IFN‐α‐induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA‐binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN‐α‐induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN‐induced apoptosis in virus‐associated tumors.

The ubiquitous Epstein-Barr virus (EBV) is associated with several human tumors, which include lymphoid and epithelial malignancies. It is known that EBV persistently infects the memory B cell pool of healthy individuals by activating growth and survival signaling pathways that can contribute to B cell lymphomagenesis. Although the monoclonal proliferation of EBV-infected cells can be observed in epithelial tumors, such as nasopharyngeal carcinoma and EBV-associated gastric carcinoma, the precise role of EBV in the carcinogenic progress is not fully understood. This review features characteristics and current understanding of EBV-associated gastric carcinoma. EBV-associated gastric carcinoma comprises almost 10% of all gastric carcinoma cases and expresses restricted EBV latent genes (Latency I). Firstly, definition, epidemiology, and clinical features are discussed. Then, the route of infection and carcinogenic role of viral genes are presented. Of particular interest, the association with frequent genomic CpG methylation and role of miRNA for carcinogenesis are topically discussed. Finally, the possibility of therapies targeting EBV-associated gastric carcinoma is proposed.

Epstein–Barr virus (EBV) is an exceptionally successful human viral pathogen maintained as a licensed, plasmid replicon in proliferating cells. We have measured the distributions of EBV‐derived plasmids in single live cells throughout the cell cycle in the absence of selection and confirmed the measured rates of duplication and partitioning computationally and experimentally. These analyses have uncovered a striking, non‐random partitioning for this minimalist plasmid replicon and revealed additional properties of it and its host cells: (1) 84% of the plasmids duplicate during each S phase; (2) all duplicated plasmids are spatially colocalized as pairs, a positioning that is coupled to their non‐random partitioning; (3) each clone of cells requires a certain threshold number of plasmids per cell for its optimal growth under selection; (4) defects in plasmid synthesis and partitioning are balanced to yield wide distributions of plasmids in clonal populations of cells for which the plasmids provide a selective advantage. These properties of its plasmid replicon underlie EBV's success as a human pathogen.